Preparation Method of Activated or Solubilized Mutated Enzyme

ABSTRACT

A method for producing an active-form mutant enzyme, by specifying a protein of which a native form exhibits an enzyme activity but which has 10% or less enzyme activity of the native form when a gene of the protein is expressed to provide an inactive-form enzyme; determining a sequence conservation of amino acid residues in an amino acid sequence of the inactive-form enzyme and specifying amino acid residue(s) for which sequence conservation in the inactive-form enzyme is lower than sequence conservation of other amino acid(s) of the same residue; preparing a gene having a base sequence that codes for the amino acid sequence of the inactive-form enzyme in which at least one said specified amino acid residue is substituted by another amino acid with a higher sequence conservation; and expressing the gene to obtain an enzyme that exhibits an enzyme activity of the native form protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 15/511,706 filed on Mar. 16, 2017, currently pending, which, inturn, is a 371 continuation of International Application no.PCT/JP2016/067392 filed on Jun. 10, 2016, which, in turn, claimspriority to Japanese Patent Application No. 2015-117808 filed on 10 Jun.2015, the disclosures of which are entirely incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to a method for producing an active-formmutant enzyme and a novel active-form mutant enzyme and a method forproducing a soluble mutant protein. More specifically, the presentinvention relates to a method for obtaining an active-form mutant enzymeby mutagenesis to an enzyme that is expressed in a heterologousexpression system as an inactive-form enzyme, the method includingselecting an effective mutation site and an amino acid after mutation,and relates to a novel active-form mutant enzyme obtained by the method.The present invention further relates to a method for obtaining asoluble mutant protein by mutagenesis to a protein that is expressed ina heterologous expression system as an insoluble-form protein, themethod including selecting an effective mutation site and an amino acidafter mutation.

BACKGROUND ART

There are many cases when an enzyme protein gene is expressed in aheterologous expression system, the protein is not expressed as anactive-form enzyme and this is the biggest problem in a large-scaleexpression of enzyme protein by a heterologous host. In order to solvethe problem, various approaches have been taken. For example, in orderto express an active-form enzyme in a host Escherichia coli, cellculture conditions are examined (PTL 1), and the following methods arementioned: methods in which proteins are co-expressed with molecularchaperones which allow formation of correct conformation (PTL 2),methods in which proteins are expressed as fusion proteins with signalpeptides or tags that improve solubility (PTL 3 and PTL 4) and methodsin which proteins which form inclusion bodies are unfolded with adenaturing agent and the like and refolded into correct conformation(PTL 5, PTL 6 and PTL 7). In addition, methods in which, without usingE. coli, enzyme genes having the same amino acid sequences as the wildtype are expressed in yeast, insect or animal cultured cells (NPL 1 andNPL 2) and cell-free translation systems in which entire transcriptionto translation of genes is carried out in vivo (PTL 8) have also beendeveloped.

However, there are many proteins for which inclusion bodies are noteliminated by co-expression with chaperone genes and other methods alsohave issues such as complicated procedures and high cost. Therefore,there is still a need for a new technique which allows expression of anenzyme gene as an active-form enzyme by the simplest and the most costeffective way.

Until now, a reported example of expression of a heterologous enzyme inE. coli as an active-form mutant enzyme is expression of a solubleprotein from a plant-derived hydroxynitrile lyase gene containing randommutations (PTL 9). There is also a report on a method in which a genesequence of a target protein is subjected to random mutation followed bythe addition of a reporter protein so as to select a sequence that isexpressed as an active-form mutant enzyme (NPL 3). However, there is noreport on a method that allows specifying a mutation site of a gene or amutated amino acid that allows expression of a protein as an active-formmutant enzyme.

When genes of various proteins including enzymes are expressed inheterologous expression systems, the proteins are not always expressedas soluble proteins. This is a problem in a large-scale expression ofvarious proteins by a heterologous host. In order to solve the problem,there have been known co-expression with molecular chaperone proteins(NPL 4), expression as fusion proteins with maltose-binding proteins(NPL 5) and the like.

CITATION LIST Patent Literature

-   [PTL 1] Japanese Patent Application Laid-open No. 2012-44888-   [PTL 2] Japanese Patent Application Laid-open No. H11-9274-   [PTL 3] Japanese Patent Application Laid-open No. 2012-116816-   [PTL 4] Japanese Patent Application Laid-open No. 2012-179062-   [PTL 5] Japanese Patent Application Laid-open No. H11-335392-   [PTL 6] Japanese Patent Application Laid-open No. 2011-46686-   [PTL 7] Japanese Translation of PCT Application No. 2001-503614-   [PTL 8] Japanese Patent Application Laid-open No. 2004-105070-   [PTL 9] WO 2006/041226

Non Patent Literature

-   [NPL 1] Meth. In Molecular Biol., Protocols 103 (1998)-   [NPL 2] J. Biol. Chem., 264, pp. 8222-8229 (1989)-   [NPL 3] Microb. Cell Fact., 4, pp. 1-8 (2005)-   [NPL 4] Appl. Environ. Micorobiol., 64, pp. 1694-1699 (1998)-   [NPL 5] Protein science, 8, pp. 1669-1674 (1999)-   [NPL 6] J. Mol. Biol., 157, pp. 105-132 (1982)-   [NPL 7] Sci. Rep., 5 doi: 10.1038/srep08193 (2015)

SUMMARY OF INVENTION Technical Problem

As described above, co-expression with chaperone genes and use of yeastand animal cells, for example, have been developed as method forexpressing active-form mutant enzymes of proteins which are otherwiseexpressed as inactive-form enzymes in heterologous expression systems.However, the developed methods have such problems that procedures arecomplicated or the methods are expensive. In addition, only limitedtypes of enzymes could be soluble by applying the methods and thus it isdifficult to widely apply the methods to enzymes for industrialapplications. Although some enzymes are reported to be expressed asactive-form mutant enzymes by mutagenesis, there has been no knownmethod that allows effective identification of a gene mutation site or amutated amino acid that is efficacious for expressing a wide range ofenzymes as active-form mutant enzymes. Similar to inactive-form enzymes,there has been no known method that allows effective identification of agene mutation site or a mutated amino acid that is efficacious forexpressing a soluble form of a protein which is otherwise expressed asan insoluble protein in a heterologous expression system.

Thus, an object of the present invention is to provide a method forexpressing an enzyme as an active-form mutant enzyme, the enzyme beingnot expressed as an active-form enzyme or expressed as an active enzymeat a minute amount in a heterologous expression system, includingselecting an effective mutation site and an amino acid after mutation.Another object of the present invention is to provide a novelactive-form mutant enzyme obtained by the method.

Another object of the present invention is to provide a method forexpressing a protein as a soluble mutant protein, the protein being notexpressed as a soluble protein or expressed as a soluble protein at aminute amount in a heterologous expression system, including selectingan effective mutation site and an amino acid after mutation.

Solution to Problem

In order to achieve the above objects, the inventors of the presentinvention investigated the relationship between mutation sites of enzymegenes and expression of active-form mutant enzyme genes in heterologousexpression systems and found that (1) an active-form mutant enzyme canbe expressed by substituting a hydrophobic amino acid that is present ina hydrophilic domain of an α-helix structural region of an enzymeprotein to a certain hydrophilic amino acid or by substituting ahydrophilic amino acid that is present in a hydrophobic domain of theregion to a certain hydrophobic amino acid and/or (2) an active-formmutant enzyme can be expressed by obtaining an amino acid sequence inwhich at least one amino acid with relatively low conservation to anamino acid with higher conservation than said amino acid, and (3) themethods described above are versatile methods for preparing anactive-form mutant enzyme which can be applied to various wild typeenzymes that are inactive in heterologous expression systems. Theinventors of the present invention thus completed the present invention.

The inventors of the present invention introduced mutations to enzymegenes derived from microorganisms, plants and animals which, inheterologous expression systems such as E. coli, are not expressed asactive enzymes or, even if expressed, the expression level of which islow, and then investigated by combining determination of proteinexpression by electrophoresis and activity assay of the target enzyme.The inventors of the present invention thereby found that the mutationssites in proteins exhibiting activities are “a hydrophilic amino acidthat is present in a hydrophobic domain or a hydrophobic amino acid thatis present in a hydrophilic domain”. The inventors of the presentinvention then analysed the mutation sites in detail on a secondarystructure predicting programme and revealed that the mutation sites maybe in “an α-helix region of an enzyme protein”. The inventors of thepresent invention further revealed that expression of active-form mutantenzymes has a common feature of “mutation of a hydrophilic amino acidthat is present in a hydrophobic domain of an α-helix that is present inan enzyme protein or a hydrophobic amino acid that is present in ahydrophilic domain”. The inventors of the present invention alsoanalysed in detail by dividing α-helix regions obtained by secondarystructure prediction into a hydrophilic domain and a hydrophobic domainby Edmundson wheel plot and found that a mutant enzyme exhibitingactivity may be obtained by substituting a hydrophilic amino acid thatis present in a hydrophobic domain to a hydrophobic amino acid or bysubstituting a hydrophobic amino acid that is present in a hydrophilicdomain to a hydrophilic amino acid and that there is certainrelationship between the mutation site of an amino acid (gene) andexpression of an active-form mutant enzyme.

Although the above technique is to express an active-form mutant enzymegene only by focusing the amino acid sequence of the target protein, theinventors of the present invention found that when, separate from theabove, amino acid sequences similar to that of the target protein areknown, it is possible to obtain an active-form mutant protein with highprobability and thus efficiently prepare an active-form mutant enzyme byfocusing the conservation in the sequence identity of the similar aminoacid sequences and substituting an amino acid having low conservation toan amino acid having relatively high conservation.

Although the above technique utilizes the conservation in the sequenceidentity of similar amino acid sequences, the inventors of the presentinvention further revealed that the technique may be further applied toa candidate amino acid to be mutated in an α-helix region of an enzymeprotein to more efficiently prepare an active-form mutant enzyme.

The inventors of the present invention also revealed that the abovetechnique may be applied not only for preparation of an active-formmutant enzyme but also for obtaining a soluble mutant protein of whichnative form is expressed as an insoluble protein in a heterologousexpression system.

The present invention is as follows:

[1]

A method for producing an active-form mutant enzyme comprising:expressing, in a heterologous expression system, a gene having a basesequence that codes for an amino acid sequence in which: at least onehydrophobic amino acid that is present in a hydrophilic domain of anα-helix structure region is substituted (to an amino acid with higherhydrophilicity or lower hydrophobicity than the amino acid to besubstituted) and/or at least one hydrophilic amino acid that is presentin a hydrophobic domain of the α-helix structure region is substituted(to an amino acid with higher hydrophobicity or lower hydrophilicitythan the amino acid to be substituted),

wherein the α-helix structure region is in an amino acid sequence of aprotein of which native form exhibits an enzyme activity but whichexhibits no enzyme activity or a feeble enzyme activity when expressinga gene of the protein in a heterologous expression system (hereinafter“an inactive-form enzyme); andselecting a protein (hereinafter “an active-form mutant enzyme”) thatexhibits the same sort of enzyme activity as that obtained in the nativeform.[2]

The producing method according to [1], wherein the method includes:

(1) specifying a protein of which native form exhibits an enzymeactivity but which exhibits no enzyme activity or a feeble enzymeactivity when expressing a gene of the protein in a heterologousexpression system (hereinafter “an inactive-form enzyme”);(2a) specifying an α-helix structure region of the inactive-form enzymespecified in step (1), specifying a hydrophilic domain and/orhydrophobic domain of the specified α-helix structure region, andspecifying a hydrophobic amino acid that is present in the hydrophilicdomain and/or a hydrophilic amino acid that is present in thehydrophobic domain;(3a) preparing a gene having a base sequence that codes for an aminoacid sequence in which at least one hydrophobic amino acid that ispresent in the hydrophilic domain of the α-helix structure region in theamino acid sequence of the inactive-form enzyme is substituted (to anamino acid with higher hydrophilicity or lower hydrophobicity than theamino acid to be substituted) and/or at least one hydrophilic amino acidthat is present in the hydrophobic domain of the α-helix structureregion is substituted (to an amino acid with higher hydrophobicity orlower hydrophilicity than the amino acid to be substituted); and(4a) expressing, in a heterologous expression system, the gene havingthe base sequence prepared in step (3a) to obtain a protein andselecting from the obtained proteins, a protein (hereinafter “anactive-form mutant enzyme”) that exhibits the same sort of enzymeactivity as that obtained in the native form.[3]

A method for producing a soluble mutant protein comprising:

expressing, in a heterologous expression system, a gene having a basesequence that codes for an amino acid sequence in which:at least one hydrophobic amino acid that is present in a hydrophilicdomain of an α-helix structure region is substituted (to an amino acidwith higher hydrophilicity or lower hydrophobicity than the amino acidto be substituted) and/or at least one hydrophilic amino acid that ispresent in a hydrophobic domain of the α-helix structure region issubstituted (to an amino acid with higher hydrophobicity or lowerhydrophilicity than the amino acid to be substituted),wherein the α-helix structure region is in an amino acid sequence of aprotein of which native form is a soluble protein but which becomesinsoluble when expressing a gene of the protein in a heterologousexpression system (hereinafter “an insoluble-form protein); andselecting a protein (hereinafter “a soluble mutant protein”) that issoluble.[4]

The producing method according to [3], wherein the method includes:

(1) specifying a protein of which native form is a soluble protein butwhich becomes insoluble when expressing a gene of the protein in aheterologous expression system (hereinafter “an insoluble-formprotein”);(2a) specifying an α-helix structure region of the insoluble-formprotein specified in step (1), specifying a hydrophilic domain and/orhydrophobic domain of the specified α-helix structure region, andspecifying a hydrophobic amino acid that is present in the hydrophilicdomain and/or a hydrophilic amino acid that is present in thehydrophobic domain;(3a) preparing a gene having a base sequence that codes for an aminoacid sequence in which at least one hydrophobic amino acid that ispresent in the hydrophilic domain of the α-helix structure region in theamino acid sequence of the insoluble-form protein is substituted (to anamino acid with higher hydrophilicity or lower hydrophobicity than theamino acid to be substituted) and/or at least one hydrophilic amino acidthat is present in the hydrophobic domain of the α-helix structureregion is substituted (to an amino acid with higher hydrophobicity orlower hydrophilicity than the amino acid to be substituted); and(4a) expressing, in a heterologous expression system, the gene havingthe base sequence prepared in step (3a) to obtain a protein andselecting from the obtained proteins, a protein (hereinafter “a solublemutant protein”) that is soluble.[5]

The producing method according to [3] or [4], wherein being the solublemutant protein is judged from an amount of soluble protein in an extractafter heterologous expression.

[6]

The producing method according to [5], wherein the soluble mutantprotein is defined as a mutant protein of which amount of the solubleprotein in the extract after heterologous expression is higher than anamount of the soluble protein in an extract after heterologousexpression of the native form protein.

[7]

The producing method according to [2] or [4], wherein in the step (2a)the hydrophilic domain and/or hydrophobic domain is specified by drawinga helical wheel of the α-helix structure region by using a secondarystructure predicting method, aligning amino acids at positions 1, 5, 2,6, 3, 7 and 4 on the helical wheel in this order to form a sequence andrepeating the procedure to form at least two amino acid sequences eachof which has 7 amino acids (it is sufficient that the second sequencehas 5 or more amino acids);

in the amino acid sequence region where at least two sequences arealigned, defining a row in which the sum of the hydropathy index of theamino acids therein is 0 or more as a hydrophobic row and defining a rowin which the sum of the hydropathy index of the amino acids therein isless than 0 as a hydrophilic row; anddefining a bunch of 3 or 4 consecutive hydrophobic rows as thehydrophobic domain and defining a bunch of 4 or 3 consecutivehydrophilic rows as the hydrophilic domain (provided that the sum of thehydropathy index of any one row that is internal to 4 hydrophobic rowsin the hydrophobic domain may be less than 0 and the sum of thehydropathy index of any one row that is internal to 4 hydrophilic rowsin the hydrophilic domain may be 0 or more).[8]

The producing method according to [2], [4] or [7], wherein in step (2a),an amino acid among amino acids in the hydrophilic domain, having thehydropathy index of 0 or more is specified as a hydrophobic amino acidand an amino acid, among amino acids in the hydrophobic domain, havingthe hydropathy index of less than 0 is specified as a hydrophilic aminoacid.

[9]

The producing method according to any one of [2], [4], [7] and [8],wherein in the step (3a), the hydrophobic amino acid that is present inthe hydrophilic domain is substituted to an amino acid with lowerhydropathy index than the amino acid to be substituted, and

the hydrophilic amino acid that is present in the hydrophobic domain issubstituted to an amino acid with higher hydropathy index than the aminoacid to be substituted.[10]

The producing method according to [2], [4] or [7], wherein in step (3a),the hydrophobic amino acid that is present in the hydrophilic domain issubstituted to an amino acid with lower hydropathy index than the aminoacid to be substituted and with a hydropathy index of less than 0, and

the hydrophilic amino acid that is present in the hydrophobic domain issubstituted to an amino acid with higher hydropathy index than the aminoacid to be substituted and with a hydropathy index of 0 or more.[11]

The producing method according to any one of [2], [4] and [7] to [10],wherein in step (3a), the amino acid is substituted to an amino acidwith higher conservation in sequence identity than the amino acid to besubstituted.

[12]

The producing method according to any one of [2], [4] and [7] to [11],wherein in the step (3a), the amino acid substitution is carried out forany of a plurality of amino acids and in step (4a), the active-formmutant enzyme or the soluble mutant protein is selected from a pluralityof proteins in which any of the amino acids are substituted.

[13]

A method for producing an active-form mutant enzyme,

comprising expressing, in a heterologous expression system, a genehaving a base sequence that codes for an amino acid sequence of aprotein of which native form exhibits an enzyme activity but whichexhibits no enzyme activity or a feeble enzyme activity when expressinga gene of the protein in a heterologous expression system (hereinafter“an inactive-form enzyme”), wherein in the amino acid sequence, at leastone amino acid with relatively low conservation is substituted to anamino acid with higher conservation than said amino acid; andobtaining a protein (hereinafter “an active-form mutant enzyme”) thatexhibits the same sort of enzyme activity as that obtained in the nativeform.[14]

The producing method according to [13], comprising:

(1) specifying a protein of which native form exhibits an enzymeactivity but which exhibits no enzyme activity or a feeble enzymeactivity when expressing a gene of the protein in a heterologousexpression system (hereinafter “an inactive-form enzyme”);(2b) determining a conservation in sequence identity of at least someamino acids in an amino acid sequence of the inactive-form enzymespecified in step (1) and specifying an amino acid with relatively lowconservation;(3b) preparing a gene having a base sequence that codes for an aminoacid sequence in which at least one amino acid with relatively lowconservation is substituted to an amino acid with higher conservationthan said amino acid; and(4b) expressing, in a heterologous expression system, the gene havingthe base sequence prepared in step (3b) to obtain a protein andselecting from the obtained proteins, a protein (hereinafter “anactive-form mutant enzyme”) that exhibits the same sort of activity asthat obtained in the native form.[15]

A method for producing a soluble mutant protein, comprising expressing,in a heterologous expression system, a gene having a base sequence thatcodes for an amino acid sequence of a protein of which native form is asoluble protein but which becomes insoluble when expressing a gene ofthe protein in a heterologous expression system (hereinafter “aninsoluble-form protein”), wherein in the amino acid sequence, at leastone amino acid with relatively low conservation is substituted to anamino acid with higher conservation than said amino acid; and obtaininga protein (hereinafter “a soluble mutant protein”) that is soluble.

[16]

The producing method according to [15], comprising:

(1) specifying a protein of which native form is a soluble protein butwhich becomes insoluble when expressing a gene of the protein in aheterologous expression system (hereinafter “an insoluble-formprotein”);(2b) determining a conservation in sequence identity of at least someamino acids in an amino acid sequence of the insoluble-form proteinspecified in step (1) and specifying an amino acid with relatively lowconservation;(3b) preparing a gene having a base sequence that codes for an aminoacid sequence in which at least one amino acid with relatively lowconservation is substituted to an amino acid with higher conservationthan said amino acid; and(4b) expressing, in a heterologous expression system, the gene havingthe base sequence prepared in step (3b) to obtain a protein andselecting from the obtained proteins, a soluble mutant protein.[17]

The producing method according to [15] or [16], wherein being thesoluble mutant protein is judged from an amount of the soluble proteinin an extract after heterologous expression.

[18]

The producing method according to [17], wherein the soluble mutantprotein is defined as a mutant protein of which amount of the solubleprotein in the extract after heterologous expression is higher than anamount of soluble protein in an extract after heterologous expression ofthe native form protein.

[19]

The producing method according to [14] or [16], wherein the amino acidsequence for which the conservation in sequence identity is determinedin step (2b) is selected from an amino acid sequence of an α-helixstructure region of the inactive-form enzyme or the insoluble-formprotein specified in step (1).

[20]

The producing method according to [14], [16] or [19], wherein thesubstituting amino acid is any one of three amino acids with the highestconservation in sequence identity.

[21]

The producing method according to any one of [14], [16], [19] and [20],wherein in step (3b), the amino acid substitution is carried out for aplurality of amino acids and in the step (4b), the active-form mutantenzyme or the soluble mutant protein is selected from proteins in whicha plurality of amino acids are substituted.

[22]

The producing method according to any one of [1] to [21], wherein theheterologous expression system is an Escherichia coli expression system,a yeast expression system, a Brevibacillus expression system, aCorynebacterium expression system, an Aspergillus expression system, aninsect cell expression system, an Actinomyces expression system, a plantcell expression system, an animal cell expression system or a cell-freeprotein synthetic system.

[23]

The producing method according to any one of [1] to [22], wherein theactive-form mutant enzyme has an enzyme activity value in the range of 2times to infinity of the inactive-form enzyme.

[24]

An active-form mutant mandelonitrile oxidase having an amino acidsequence shown in SEQ ID NO: 2, wherein:

valine at the position 444 has been substituted to threonine, serine,tyrosine, histidine, glutamic acid, glutamine, aspartic acid,asparagine, lysine or arginine and/orvaline at the position 455 has been substituted to glutamic acid,glutamine, aspartic acid, asparagine, lysine or arginine.[25]

An active-form mutant arginine decarboxylase having an amino acidsequence shown in SEQ ID NO: 3, wherein:

valine at the position 261 has been substituted to threonine, serine,glutamic acid, aspartic acid, asparagine, lysine or arginine and/orarginine at the position 430 has been substituted to valine, leucine oralanine, and leucine at the position 435 has been substituted tohistidine, glutamic acid, glutamine, aspartic acid, asparagine orlysine.[26]

An active-form mutant ornithine decarboxylase having an amino acidsequence shown in SEQ ID NO: 7, wherein:

lysine at the position 117 has been substituted to leucine and/orleucine at the position 176 has been substituted to glutamic acid.[27]

An active-form mutant luciferase having an amino acid sequence shown inSEQ ID NO: 5, wherein:

isoleucine at the position 80 has been substituted to lysine and/oralanine at the position 177 has been substituted to aspartic acid.[28]

An active-form mutant glutamate dehydrogenase having an amino acidsequence shown in SEQ ID NO: 9, wherein:

valine at the position 174 has been substituted to aspartic acid,lysine at the position 257 has been substituted to tyrosine and/orleucine at the position 261 has been substituted to glutamic acid.[29]

A method for producing an active-form mutant enzyme comprisingexpressing a gene that codes for the amino acid sequence according toany one of [24] to [28] in an Escherichia coli expression system andobtaining a protein.

[30]

The producing method according to [29], wherein the active-form mutantenzyme is an active-form mutant mandelonitrile oxidase, an active-formmutant arginine decarboxylase, an active-form mutant ornithinedecarboxylase, an active-form mutant luciferase or an active-form mutantglutamate dehydrogenase.

[31]

The producing method according to any one of [3] to [22], wherein theinsoluble-form protein is one protein selected from the group consistingof an enzyme, a cytokine, a haemoglobin and a myoglobin.

[32]

The method according to any one of [3] to [22], wherein theinsoluble-form protein is one protein selected from the group consistingof IFN-γ, IL-2, IFNβ and human growth hormone.

Advantageous Effects of Invention

According to the present invention, various enzymes which areinactive-form enzymes in a heterologous expression system may beexpressed as active-form mutant enzymes in the heterologous expressionsystem which are mutant proteins in which a hydrophobic amino acid thatis present in a hydrophilic domain of an α-helix structure region in theenzyme proteins is substituted to more hydrophilic amino acid or mutantproteins in which a hydrophilic amino acid that is present in ahydrophobic domain of the region is substituted to more hydrophobicamino acid.

Further, according to the present invention, by focusing on theconservation in sequence identity of similar amino acid sequences, anactive-form mutant enzyme may be expressed in a heterologous expressionsystem which is a mutant protein in which an amino acid with lowconservation is substituted to an amino acid with higher conservation.

According to the present invention, various proteins which are insolublein a heterologous expression system may be expressed as soluble mutantproteins in the heterologous expression system which are mutant proteinsin which a hydrophobic amino acid that is present in a hydrophilicdomain of an α-helix structure region in the proteins is substituted tomore hydrophilic amino acid or mutant proteins in which a hydrophilicamino acid that is present in a hydrophobic domain of the region issubstituted to more hydrophobic amino acid.

Further, according to the present invention, by focusing on theconservation in sequence identity of similar amino acid sequences, asoluble mutant protein may be expressed in a heterologous expressionsystem which is a mutant protein in which an amino acid with lowconservation is substituted to an amino acid with higher conservation.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a graphical image of secondary structure prediction ofmandelonitrile oxidase and FIG. 1B is a helical wheel of an α-helix(RVDIDTMVRGVHVALNFG) containing a mutation site of valine at theposition 455 and hydrophilic and hydrophobic domains and the mutationsite, valine at the position 455.

FIG. 2A is a helical wheel projection (left panel) and the sequencesobtained by aligning amino acids 1 to 18 in an amino acid sequence onthe helical wheel projection in a clockwise manner and FIG. 2B is aminoacid sequences on the helix obtained by beginning new rows every 7residues and hydrophilic and hydrophobic domains therein; the helicalwheel is believed to have 3.6 residues per period, the eighth residuereturns to the position close to the first residue and the rest of theresidues describe a similar trajectory; an open circle represents ahydrophilic amino acid and a filled circle represents a hydrophobicamino acid; and as apparent from the left panel, the domain containinghydrophilic amino acids 2, 5, 6, 12, 13 and 16 is taken as a hydrophilicdomain, the domain containing hydrophobic amino acids 1, 3, 4, 7, 8, 10,14, 15, 17 and 18 is taken as a hydrophobic domain and it is consideredthat hydrophobic amino acid 9 in the hydrophilic domain and hydrophilicamino acid 11 in the hydrophobic domain are the sites at which mutationsare introduced.

FIG. 3 is an illustration relating to the method (the producing methodof the first embodiment and the producing method of the secondembodiment of the present invention) for specifying a residue which maypossibly allow expression of an active-form mutant enzyme.

FIG. 4 shows oxidase activity (U/ml) and amino acid sequenceconservation (%) of enzymes obtained by substituting valine at theposition 455 of mandelonitrile oxidase to other 19 amino acids; barsindicate oxidase activity and open circles show amino acid sequenceconservation; and the letters are as follows: A: alanine, C: cysteine,D: aspartic acid, E: glutamic acid, F: phenylalanine, G: glycine, H:histidine, I: isoleucine, K: lysine, L: leucine, M: methionine, N:asparagine, P: proline, Q: glutamine, R: arginine, S: serine, T:threonine, V: valine, W: tryptophan and Y: tyrosine.

FIG. 5 shows oxidase activity (U/ml) and amino acid sequenceconservation (%) of enzymes obtained by substituting valine at theposition 444 of mandelonitrile oxidase to other 19 amino acids; barsindicate oxidase activity and open circles show amino acid sequenceconservation; and the letters are as follows: A: alanine, C: cysteine,D: aspartic acid, E: glutamic acid, F: phenylalanine, G: glycine, H:histidine, I: isoleucine, K: lysine, L: leucine, M: methionine, N:asparagine, P: proline, Q: glutamine, R: arginine, S: serine, T:threonine, V: valine, W: tryptophan and Y: tyrosine.

FIG. 6A shows a helical wheel obtained with R443 to G460 ofmandelonitrile oxidase according to the drawing method shown in FIGS.2A-2B (secondary structure predicting method); and FIG. 6B is a linearrepresentation of the helical wheel in FIG. 6A.

FIG. 6C shows the linear representation of the helical wheel togetherwith the hydropathy index of each amino acid and the sum of each row.

FIG. 7A is a graphical image of secondary structure prediction ofarginine decarboxylase and FIG. 7B shows a helical wheel of 8th and 13thα-helices from the N-terminal and hydrophilic and hydrophobic domainsand mutation sites.

FIG. 7C shows the linear representation of the helical wheel togetherwith the hydropathy index of each amino acid and the sum of each row.

FIG. 8 shows decarboxylase activity (U/ml) and amino acid sequenceconservation (%) of enzymes obtained by substituting valine at theposition 261 of arginine decarboxylase to other 19 amino acids; barsindicate oxidase activity and open circles show amino acid sequenceconservation; and the letters are as follows: A: alanine, C: cysteine,D: aspartic acid, E: glutamic acid, F: phenylalanine, G: glycine, H:histidine, I: isoleucine, K: lysine, L: leucine, M: methionine, N:asparagine, P: proline, Q: glutamine, R: arginine, S: serine, T:threonine, V: valine, W: tryptophan and Y: tyrosine.

FIG. 9 shows decarboxylase activity (U/ml) and amino acid sequenceconservation (%) of enzymes obtained by substituting arginine at theposition 430 of arginine decarboxylase to other 19 amino acids; barsindicate oxidase activity and open circles show amino acid sequenceconservation; and the letters are as follows: A: alanine, C: cysteine,D: aspartic acid, E: glutamic acid, F: phenylalanine, G: glycine, H:histidine, I: isoleucine, K: lysine, L: leucine, M: methionine, N:asparagine, P: proline, Q: glutamine, R: arginine, S: serine, T:threonine, V: valine, W: tryptophan and Y: tyrosine.

FIG. 10 shows decarboxylase activity (U/ml) and amino acid sequenceconservation (%) of enzymes obtained by substituting leucine at theposition 435 of arginine decarboxylase to other 19 amino acids; barsindicate oxidase activity and open circles show amino acid sequenceconservation; and the letters are as follows: A: alanine, C: cysteine,D: aspartic acid, E: glutamic acid, F: phenylalanine, G: glycine, H:histidine, I: isoleucine, K: lysine, L: leucine, M: methionine, N:asparagine, P: proline, Q: glutamine, R: arginine, S: serine, T:threonine, V: valine, W: tryptophan and Y: tyrosine.

FIG. 11A shows a secondary structure predicted for the amino acidsequence of luciferase and FIG. 11B shows a helical wheel of α-helix (1)(upper panel) and hydrophilic and hydrophobic domains and the mutationsite (lower panel).

FIG. 12 shows the luminescence value of luciferase reaction of crudeenzyme solutions of a wild type enzyme of luciferase, a mutant enzymeobtained by substituting isoleucine at the position 80 to lysine and amutant enzyme obtained by substituting alanine at the position 177 toaspartic acid.

FIG. 13 shows an image of SDS-PAGE electrophoresis of enzymes purifiedby nickel affinity chromatography of a mutant enzyme in which isoleucineat the position 80 of luciferase is substituted to lysine and a mutantenzyme in which alanine at the position 177 is substituted to asparticacid.

FIG. 14 shows a graphical image of secondary structure prediction ofornithine decarboxylase.

FIG. 15 shows a graphical image of secondary structure prediction ofglutamic acid dehydrogenase.

FIG. 16 shows the test results of the expression level of mandelonitrileoxidase; and soluble ChMOX indicates supernatant (soluble fraction) andSoluble+Insoluble ChMOX indicates the entire expression level(supernatant (soluble fraction)+pellet (insoluble fraction)).

FIG. 17 shows the test results of the expression level of soluble hGHmutant protein using hGH ELISA.

FIG. 18 shows SDS-PAGE after purification.

FIG. 19 shows the amino acid sequence of hGH (SEQ ID NO: 11).

DESCRIPTION OF EMBODIMENTS

<Producing Method of an Active-Form Mutant Enzyme (Producing Method ofthe First Embodiment)>

The method for producing an active-form mutant enzyme of the presentinvention (producing method of the first embodiment) is:

a method for producing an active-form mutant enzyme comprising:expressing, in a heterologous expression system, a gene having a basesequence that codes for an amino acid sequence in which:at least one hydrophobic amino acid that is present in a hydrophilicdomain of an α-helix structure region is substituted (to an amino acidwith higher hydrophilicity or lower hydrophobicity than the amino acidto be substituted) and/or at least one hydrophilic amino acid that ispresent in a hydrophobic domain of the α-helix structure region issubstituted (to an amino acid with higher hydrophobicity or lowerhydrophilicity than the amino acid to be substituted),wherein the α-helix structure region is in an amino acid sequence of aprotein of which native form exhibits an enzyme activity but whichexhibits no enzyme activity or a feeble enzyme activity when expressinga gene of the protein in a heterologous expression system (hereinafter“an inactive-form enzyme); andselecting a protein (hereinafter “an active-form mutant enzyme”) thatexhibits the same sort of activity as that obtained in the native form.

The method for producing the active-form mutant enzyme specificallyinclude the following steps:

(1) specifying a protein of which native form exhibits an enzymeactivity but which exhibits no enzyme activity or a feeble enzymeactivity when expressing a gene of the protein in a heterologousexpression system (hereinafter “an inactive-form enzyme”);(2a) specifying an α-helix structure region of the inactive-form enzymespecified in step (1), specifying a hydrophilic domain and/orhydrophobic domain of the specified α-helix structure region, andspecifying a hydrophobic amino acid that is present in the hydrophilicdomain and/or a hydrophilic amino acid that is present in thehydrophobic domain;(3a) preparing a gene having a base sequence that codes for an aminoacid sequence in which at least one hydrophobic amino acid that ispresent in the hydrophilic domain of the α-helix structure region in theamino acid sequence of the inactive-form enzyme is substituted (to anamino acid with higher hydrophilicity or lower hydrophobicity than theamino acid to be substituted) and/or at least one hydrophilic amino acidthat is present in the hydrophobic domain of the α-helix structureregion is substituted (to an amino acid having higher hydrophobicity orlower hydrophilicity than the amino acid to be substituted); and(4a) expressing, in a heterologous expression system, the gene havingthe base sequence prepared in step (3a) to obtain a protein andselecting from the obtained proteins, a protein (“an active-form mutantenzyme”) that exhibits the same sort of activity as that obtained in thenative form.

Step (1)

In step (1), a protein of which native form exhibits an enzyme activitybut which exhibits no enzyme activity or a feeble enzyme activity whenexpressing a gene of the protein in a heterologous expression system isspecified. Such a protein is referred to as an inactive-form enzyme. Theinactive-form enzyme as used herein also encompasses an enzyme whichexhibits activity when expressed in a certain heterologous expressionsystem but is inactive in other heterologous expression systems. Thesource or type of the inactive-form enzyme is not particularly limited.

The protein (hereinafter also referred to as “a target protein”) whichis a subject of the present invention and of which native form exhibitsan enzyme activity is not particularly limited and examples thereofinclude a protein of microorganism origin, a protein of animal originand a protein of plant origin. The type of the inactive-form enzyme isnot particularly limited and examples thereof include oxidoreductases,transferases, hydrolases, isomerases, lyases and ligases.

The heterologous expression system refers to, when the system producinga target protein is an organism, an expression system employing a hostthat is different from the organism producing the target protein, or acell-free protein synthetic system. The type of the expression systememploying a different host or the type of the cell-free proteinsynthetic system are not particularly limited. When the organismproducing a target protein is a cell-free protein synthetic system, theheterologous expression system refers to an expression system employingany host or a cell-free protein synthetic system which is of a differenttype from the cell-free protein synthetic system producing the targetprotein. The expression system employing a host may include thosegenerally used in genetic engineering without limitation and examplesthereof include an E. coli expression system, a yeast expression system,a Brevibacillus expression system, a Corynebacterium expression system,an Aspergillus expression system, an insect cell expression system, anActinomyces expression system, a plant cell expression system and ananimal cell expression system. Examples of the cell-free proteinsynthetic system include human cultured cells, rabbit reticulocytes,insect cultured cells, wheat germ, an extreme thermophile Thermusthermophilus, a hyperthermophile archaea Thermococcus kodakarensis, acell-free protein synthetic system employing an E. coli extract, areconstituted cell-free protein synthetic system PURE system and thelike.

Specifying an inactive-form enzyme in a heterologous expression systemis carried out by introducing a gene that codes for a protein of whichnative form exhibits an enzyme activity into a heterologous expressionsystem, expressing the protein in the heterologous expression system andexamining whether or not the obtained protein has the enzyme activity asexhibited by the native form and if the protein exhibits the activity,how much the activity is. Expression in a heterologous expression systemmay be carried out by a standard manner of each heterologous expressionsystem. The details are described hereinafter.

The phrase “a protein which exhibits no activity when expressing a geneof the protein in a heterologous expression system” is the case wherethe result of an activity assay of the subject enzyme is at or below thedetection limit. The phrase “a protein which exhibits a feeble activity”is the case where the activity in an activity assay of the subjectenzyme is 10% or less, preferably 5% or less and more preferably 1% orless of the enzyme activity exhibited by the protein in the native form.

Step (2a)

Step (2a) is the step of specifying an α-helix structure region of theinactive-form enzyme specified in step (1) (substep 1), specifying ahydrophilic domain and/or hydrophobic domain of the specified α-helixstructure region (substep 2) and specifying a hydrophobic amino acidthat is present in the hydrophilic domain and/or a hydrophilic aminoacid that is present in the hydrophobic domain (substep 3). Step (2a)includes 3 substeps.

In substep 1, an α-helix structure region of the inactive-form enzymespecified in step (1) is specified by, for example, a secondarystructure predicting method. Specifying an α-helix structure region ofthe inactive-form enzyme specified in step (1) by a secondary structurepredicting method may be carried out by predicting the secondarystructure using, for example, a gene analysis software such as a geneticinformation processing software GENETYX or a secondary structurepredicting programme such as PSIPRED(http://bioinf.cs.ucl.ac.uk/psipred/) (FIG. 1A). In FIG. 1A, amino acidsS15 to N22, L49 to F50, G52 to Q53, S91 to V100, F107 to A112, P125 toK129, V159 to E170, S264 to L270, P276 to L279, D324 to D332, N389 toY395, R443 to G460, K463 to K467, D488 to H497 and N551 to W569correspond to an α-helix region.

All amino groups in amino acids constituting a back bone of an α-helixform hydrogen bonds with carboxyl groups of amino acids which are 4residues away. The number of amino acids constituting an α-helix regionis not limited; however, in view of finding a hydrophobic domain and ahydrophilic domain in the following substep 2, the number is preferably7 residues or more and more preferably 12 residues or more.

Substep 2 is the substep of specifying a hydrophilic domain and/orhydrophobic domain of the specified α-helix structure region. Specifyinga hydrophilic domain and/or a hydrophobic domain may be carried out bydrawing a helical wheel of the α-helix structure region. Helical wheelsmay be generated by using pepwheel(http://emboss.bioinformatics.nl/cgi-bin/emboss/pepwheel) and the like(FIG. 1B). A helical wheel such as the one in FIG. 1B represents thetype and position of amino acids in an α-helix. In the presentinvention, it is preferable to draw a helical wheel with 7 residues percycle as shown in FIG. 2A, then align amino acids belonging to the helixin the helical wheel shown in FIG. 2A in order of 1→5→2→6→3→7→4, begin anew row at the eighth residue which is regarded as 1 as above and draw alinear representation of a helical wheel (FIG. 2B), thereby specifying ahydrophilic domain and/or a hydrophobic domain of the α-helix structureregion.

More specifically, the following is a preferable embodiment ofspecifying a hydrophilic domain and/or a hydrophobic domain.

(a) A helical wheel of an α-helix structure region is drawn by using asecondary structure predicting method, amino acids at positions 1, 5, 2,6, 3, 7 and 4 on the helical wheel are aligned in this order to form asequence and the procedure is repeated to form at least two amino acidsequences each of which has 7 amino acids; however, it is sufficientthat the second sequence has 5 or more amino acids. Namely, with thesecond sequence having at least 5 amino acids, a hydrophilic domainand/or a hydrophobic domain may be specified (drawing of a linearrepresentation of helical wheel).(b) In the amino acid sequence region where at least two sequences arealigned, a row in which the sum of the hydropathy index of the aminoacids therein is 0 or more is defined as a hydrophobic row and a row inwhich the sum of the hydropathy index of the amino acids therein is lessthan 0 is defined as a hydrophilic row.(c) A bunch of 3 or 4 consecutive hydrophobic rows is defined as thehydrophobic domain and a bunch of 4 or 3 consecutive hydrophilic rows isdefined as the hydrophilic domain. When a bunch of 3 consecutivehydrophobic rows is defined as a hydrophobic domain, a bunch of 4consecutive hydrophilic rows is defined as a hydrophilic domain. When abunch of 4 consecutive hydrophobic rows is defined as a hydrophobicdomain, a bunch of 3 consecutive hydrophilic rows is defined as ahydrophilic domain. The sum of the hydropathy index of any one row thatis internal to 4 hydrophobic rows in the hydrophobic domain may be lessthan 0 and the sum of the hydropathy index of any one row that isinternal to 4 hydrophilic rows in the hydrophilic domain may be 0 ormore.

The phrase in (c) “one row that is internal to 4 hydrophobic rows in thehydrophobic domain” means any one of two rows which do not directlycontact a hydrophilic domain and the phrase “one row that is internal to4 hydrophilic rows in the hydrophilic domain” means any one of two rowswhich do not directly contact a hydrophobic domain.

The hydropathy index (NPL 6) is an index representing hydrophilicity andhydrophobicity of amino acids. In the present invention, an amino acidwith a hydropathy index of less than 0 is indicated as a hydrophilicamino acid and an amino acid with a hydropathy index of 0 or more isindicated as a hydrophobic amino acid.

TABLE 1 Hydropathy index Hydropathy Amino acid index Isoleucine (I) 4.5Hydrophobic Valine (V) 4.2 amino acids Leucine (L) 3.8 Phenylalanine (F)2.8 Cysteine (C) 2.5 Methionine (M) 1.9 Alanine (A) 1.8 Glycine (G) −0.4Hydrophilic Threonine (T) −0.7 amino acids Tryptophan (W) −0.9 Serine(S) −0.8 Tyrosine (Y) −1.3 Proline (P) −1.6 Histidine (H) −3.2 Glutamicacid (E) −3.5 Glutamine (Q) −3.5 Aspartic acid (D) −3.5 Asparagine (N)−3.5 Lysine (K) −3.9 Arginine (R) −4.5

Specifying a hydrophilic domain and/or a hydrophobic domain by thismanner is more specifically described hereinafter by using the proteinsin Examples as an example.

The following description is made by using mandelonitrile oxidasederived from Chamberlinius hualinensis as an example and referring toFIGS. 6A to 6C. FIG. 6A is a helical wheel of an α-helix structureregion drawn by a secondary structure predicting method as in (a). FIG.6B is a linear representation of the helical wheel in FIG. 6A. Eachcircle represents an amino acid, an open circle represents a hydrophilicamino acid, a filled circle represents a hydrophobic amino acid and eachnumber represents the position of each amino acid. FIG. 6C shows thelinear representation of the helical wheel together with the hydropathyindex of each amino acid. For example, the amino acid R (arginine) shownon the top right has a hydropathy index of −4.5. On the left of thefigure, the sums of the hydropathy index of amino acids in each row areindicated, which are, from the top in this order, (i) 3.5, (ii) −6.7,(iii) −3.8, (iv) 3.5, (v) −1.1, (vi) 3.7 and (vii) 8.3. In (b), it isdefined that in the amino acid sequence region where at least twosequences are aligned (in this example, three sequences are aligned),the row having the sum of the hydropathy index of amino acids in the rowof 0 or more is defined as a hydrophobic row and the row having the sumof less than 0 is defined as a hydrophilic row, and thus the row (i) of3.5 is defined as a hydrophobic row, the row (ii) of −6.7 is defined asa hydrophilic row, the row (iii) of −3.8 is defined as a hydrophilicrow, the row (iv) of 3.5 is defined as a hydrophobic row, the row (v) of−1.1 is defined as a hydrophilic row, the row (vi) of 3.7 is defined asa hydrophobic row and the row (vii) of 8.3 is defined as a hydrophobicrow. In (c), it is defined that a bunch of 3 or 4 consecutivehydrophobic rows is defined as a hydrophobic domain and a bunch of 4 or3 consecutive hydrophilic rows is defined as a hydrophilic domain andwhen a bunch of 3 consecutive hydrophobic rows is defined as ahydrophobic domain, a bunch of 4 consecutive hydrophilic rows is definedas a hydrophilic domain and when a bunch of 4 consecutive hydrophobicrows is defined as a hydrophobic domain, a bunch of 3 consecutivehydrophilic rows is defined as a hydrophilic domain. According to thedefinition, the bunch of 4 consecutive rows of (ii), (iii), (iv) and (v)may be defined as a hydrophobic domain and the bunch of 3 consecutiverows of (vi), (vii) and (i) may be defined as a hydrophilic domain.Although the row (iv) of 3.5 in the hydrophilic domain is a hydrophobicrow, this row corresponds to the row that is internal to 4 hydrophilicrows (namely, the row that does not directly contact a hydrophobicdomain), and thus the row corresponds to an exception as described inthe provision where the sum of the hydropathy index of any one row thatis internal to 4 hydrophilic rows in the hydrophilic domain may be 0 ormore.

As described above, in this example, the bunch of 4 consecutive rows(ii), (iii), (iv) and (v) may be defined as a hydrophobic domain and thebunch of 3 consecutive rows (vi), (vii) and (i) may be defined as ahydrophilic domain.

Substep 3 is the substep of specifying a hydrophobic amino acid that ispresent in the hydrophilic domain and/or a hydrophilic amino acid thatis present in the hydrophobic domain. Specifying a hydrophobic aminoacid that is present in a hydrophilic domain and specifying ahydrophilic amino acid that is present in a hydrophobic domain arecarried out by using the hydropathy index. In the present invention,among amino acids in the hydrophilic domain, an amino acid with ahydropathy index of 0 or more is specified as a hydrophobic amino acidand among amino acids in the hydrophobic domain, an amino acid with ahydropathy index of less than 0 is specified as a hydrophilic aminoacid.

The hydrophilic amino acid includes threonine (T), serine (S), tyrosine(Y), histidine (H), glutamic acid (E), glutamine (Q), aspartic acid (D),asparagine (N), lysine (K), arginine (R) and glycine (G) and thehydrophobic amino acid includes isoleucine (I), valine (V), leucine (L),phenylalanine (F), cysteine (C), methionine (M) and alanine (A) (seeTable 1). As tryptophan (W) and proline (P) have a hydropathy index ofless than 0, the amino acids are classified as hydrophilic amino acids.However, in the nature of the side chains, the amino acids areclassified as hydrophobic amino acids. Therefore, it is defined thattryptophan and proline do not belong to either hydrophilic orhydrophobic amino acid and upon calculation of the sum of the hydropathyindex of amino acids in a row as described above, the hydropathy indexof both tryptophan and proline is regarded as 0 (zero). However, interms of substitution of amino acids in the step (3a), tryptophan (W)and proline (P) may be included in hydrophilic amino acids as examplesof substituting amino acids.

For example, in the example shown in FIG. 6C described above, 2 Vs(valines), G (glycine) and F (phenylalanine) are hydrophobic amino acidsthat are present in the hydrophilic domain and R (arginine) is ahydrophilic amino acid that is present in the hydrophobic domain.

Step (3a)

A gene is prepared which has a nucleic acid sequence that codes for anamino acid sequence in which at least one hydrophobic amino acid that ispresent in the hydrophilic domain of the α-helix structure region in theamino acid sequence of the inactive-form enzyme is substituted. In thiscase, the amino acid is substituted to an amino acid with higherhydrophilicity or lower hydrophobicity than the amino acid to besubstituted. Alternatively, a gene is prepared which has a nucleic acidsequence that codes for an amino acid sequence in which at least onehydrophilic amino acid that is present in the hydrophobic domain of theα-helix structure region in the amino acid sequence of the inactive-formenzyme is substituted. In this case, the amino acid is substituted to anamino acid with higher hydrophobicity or lower hydrophilicity than theamino acid to be substituted. The degree of hydrophilicity and thedegree of hydrophobicity may be determined by using the hydropathy indexdescribed above. Genes having known amino acid sequences may be preparedaccording to standard methods and the introduction of desired amino acidresidue mutations by way of a mutation introducing technique may also becarried out according to standard methods. In the present invention, amutation may be introduced at the specified residue by using, forexample, QuikChange® Lightning Site-Directed Mutagenesis Kit(manufactured by Agilent Technologies).

A hydrophobic amino acid that is present in the hydrophilic domain is,in cases when the hydropathy index is used, substituted to an amino acidwith a lower hydropathy index value than the amino acid to besubstituted. The amino acid may be still a hydrophobic amino acid evenif the amino acid has a lower hydropathy index value than the amino acidto be substituted. Further, a hydrophobic amino acid that is present inthe hydrophilic domain may be substituted to a hydrophilic amino acidwith a lower hydropathy index value than the amino acid to besubstituted and also with a hydropathy index value of less than 0.Generally, by substituting a hydrophobic amino acid that is present inthe hydrophilic domain to a hydrophilic amino acid with a lowerhydropathy index value than the amino acid to be substituted and alsowith a hydropathy index value of less than 0, the tendency of surelyobtaining an active-form mutant enzyme may be increased.

Similarly, a hydrophilic amino acid that is present in the hydrophobicdomain is substituted to an amino acid with a higher hydropathy indexvalue than the amino acid to be substituted. Further, a hydrophilicamino acid that is present in the hydrophobic domain may be substitutedto an amino acid with a higher hydropathy index value than the aminoacid to be substituted and with a hydropathy index value of 0 or more.Generally, by substituting a hydrophilic amino acid that is present inthe hydrophobic domain to an amino acid with a higher hydropathy indexvalue than the amino acid to be substituted and with a hydropathy indexvalue of 0 or more, the tendency of surely obtaining an active-formmutant enzyme may be increased.

For example, in the example shown in FIG. 6C described above, 2 Vs(valines), G (glycine) and F (phenylalanine) are hydrophobic amino acidsin the hydrophilic domain and R (arginine) is a hydrophilic amino acidthat is present in the hydrophobic domain. In Examples, 2 Vs (valines)(444 and 455) were substituted to amino acids with higher hydrophilicityor lower hydrophobicity than V. The results are indicated in FIG. 4(V455) and FIG. 5 (V444). In FIG. 4 (V455), almost half of mutantproteins exhibited enzyme activity and in FIG. 5 (V444), most of themutant proteins exhibited enzyme activity, indicating that active-formmutant enzymes were obtained.

Step (4a)

The gene having the nucleic acid sequence prepared in step (3a) isexpressed in a heterologous expression system which is the same as ordifferent from the one described above to obtain a protein having amutation introduced therein. Expression of the protein having a mutationintroduced therein in a heterologous expression system may be carriedout according to standard methods.

Steps (3a) and (4a) are more specifically described hereinbelow.

(Promoter)

In the method of the present invention, a heterologous expression hostsuch as E. coli is first prepared which harbours a gene that codes for aprotein having an amino acid sequence of a target protein in which apredetermined mutation is introduced at a predetermined position. Forexample, a heterologous expression host such as E. coli which harbours agene that codes for a protein (mutagenized target protein) in which amutation is introduced to a target protein may be prepared by providinga vector having the gene that codes for the mutagenized target proteinunder the control of an inducible promoter and introducing the vectorinto E. coli. The inducible promoter is not particularly limited and maybe any known inducible promoters. For example, an inducible promoterwhich exhibits transcriptional activity in the presence ofisopropyl-1-thio-β-D-galactoside (IPTG) may be used. Examples of such apromoter include a Trp promoter, a Lac promoter, a Trc promoter, a Tacpromoter, a T7 promoter and the like. Other promoters which exhibittranscriptional activity in the presence of other inducers than IPTG andother promoters which exhibit transcriptional activity according toculture conditions such as components in media or temperature (forexample, low temperature) may also be used as the inducible promoter.

(Vector)

The vector is not particularly limited as long as the vector may bereplicated in the heterologous expression host such as E. coli and maybe any of plasmid vectors, phage vectors and the like. Examples of thespecific vector may include vectors of pCDF series, pRSF series, pETseries and the like. For example, for an expression vector of pET(having a T7 promoter), E. coli BL21 (DE3) and JM109 (DE3) may be used.The vector may be introduced into E. coli by applying various methodsgenerally known as transformation technique. Specific methods which maybe applied include calcium phosphate transfection, electroporation,lipofection and the like. Transfection may be transient or stable.

E. coli may be the one that harbours a plasmid pLysS (BL21 (DE3) pLysS:manufactured by Invitrogen Corporation). The plasmid pLysS expresses alow level of T7 ribozyme. T7 ribozyme inhibits T7 RNA polymerase andthus can suppress expression of a mutagenized target protein beforeexpression induction by IPTG. Further, in a heterologous expression hostsuch as E. coli, a chaperone and the like may be co-expressed in orderto aid correct folding of a mutagenized target protein.

(Culture Conditions)

When culturing E. coli that harbours a gene that codes for a mutagenizedtarget protein sequence, any of a batch culture, a continuous cultureand the like may be carried out. The culture may be of a static cultureand a shake culture among which shake culture is preferred. As a medium,LB medium (5.0 g/L Yeast extract, 10.0 g/L NaCl, 10.0 g/L Tryptone) andthe like may be used. The procedure of culture is not particularlylimited as far as recombinant E. coli can be grown and examples thereofinclude culturing E. coli at 37° C. until a cell turbidity of about 0.5is obtained, adding IPTG and then culturing cells at a temperature of16° C. to 37° C. for 16 to 24 hours. In case of heterologous expressionhosts other than E. coli, well-known culture methods used for therespective hosts may be applied.

(Protein Extraction)

After the main culture, the heterologous expression host may bedisrupted to prepare a crude enzyme solution containing a mutagenizedtarget protein. In a conventional method, when a heterologous gene isexpressed at a large scale in bacteria and the like by recombinant DNAtechnique, the produced protein may form inclusion bodies which are aninsoluble substance accumulated in the cells. In the present invention,the host cells such as E. coli are collected, the cells are disrupted onan ultrasonicator or the like and separating a supernatant from a pelletincluding inclusion bodies by centrifugation to obtain the supernatant(soluble fraction) which is used as a crude enzyme solution. Themutagenized target protein obtained according to the present method isusually soluble, and thus the crude protein suspension contains themutagenized target protein having predetermined activity and function.Therefore, the obtained crude protein suspension may be directly used asa solution containing the mutagenized target protein. The mutagenizedtarget protein may alternatively be used after isolation andpurification from the obtained crude protein suspension. In this case,isolation and purification of the mutagenized protein may be carried outby a common biochemical procedure (such as ammonium sulphateprecipitation, gel chromatography, ion-exchange chromatography andaffinity chromatography) or an appropriate combination thereof. Themutagenized target protein after isolation and purification may be used,for example, in the form of a suspension in a buffer having apredetermined pH.

From mutagenized proteins expressed in the heterologous expressionsystem, a protein (active-form mutant enzyme) that exhibits the samesort of activity as that obtained in the native form is selected.Selection of the active-form mutant enzyme may be carried out asfollows. Selection may be carried out by assaying an enzyme activity inthe system in which the enzyme activity that is obtained in the nativeform can be assayed. The enzyme activity assay may appropriately becarried out by a method known for each enzyme.

For example, enzyme activity of mandelonitrile oxidase may be assayedaccording to a colorimetric assay by reaction with a substratemandelonitrile together with 4-aminoantipyrine, TOOS and peroxidase.Enzyme activity of arginine or ornithine decarboxylase may be assayedaccording to a colorimetric assay by reaction with a substrate arginineor ornithine together with an amine oxidase that acts with the oxidationreaction product, 4-aminoantipyrine, TOOS and peroxidase. Enzymeactivity of luciferase may be assayed by allowing reaction with asubstrate coelenterazine and measuring generated luminescence. Enzymeactivity of glutamate dehydrogenase may be assayed by detecting anincrement of generated NADH after addition of a substrate glutamic acidas well as NAD⁺ as an amount of change of the absorbance at 340 nm.

Based on the result of the enzyme activity assay, whether or not theactive-form mutant enzyme is obtained may be found and a mutation whichprovides the active-form mutant enzyme may be found. The active-formmutant enzyme may have, for example, an enzyme activity value that is inthe range of 2 times to infinity of the enzyme activity value of aninactive-form enzyme. When an inactive-form enzyme does not exhibit anyactivity (in cases where the result of assay is below the detectionlimit), the increase rate of the enzyme activity value is infinity andwhen an inactive-form enzyme exhibits a feeble activity, the enzymeactivity value is 2 times or more and more preferably 10 times or moreof the feeble activity.

<Method for Producing an Active-Form Mutant Enzyme (Producing Method ofthe Second Embodiment)>

The method for producing an active-form mutant enzyme of the presentinvention (producing method of the second embodiment) includes thefollowing steps:

(1) specifying a protein of which native form exhibits an enzymeactivity but which exhibits no enzyme activity or a feeble enzymeactivity when expressing a gene of the protein in a heterologousexpression system (hereinafter “an inactive-form enzyme”);(2b) determining a conservation of at least some amino acids in an aminoacid sequence of the inactive-form enzyme specified in step (1) andspecifying an amino acid with relatively low conservation;(3b) preparing a gene having a base sequence that codes for an aminoacid sequence in which at least one amino acid with relatively lowconservation is substituted to an amino acid with higher conservationthan said amino acid; and(4b) expressing, in a heterologous expression system, the gene havingthe base sequence prepared in step (3b) to obtain a protein andselecting from the obtained proteins, a protein (hereinafter “anactive-form mutant enzyme”) that has the same sort of activity as thatobtained in the native form.

Step (1) is identical to step (1) in the method for producing anactive-form mutant enzyme of the present invention (producing method ofthe first embodiment) and the description in the producing method of thefirst embodiment may be referred to.

Step (2b)

Step (2b) is the step of determining a conservation in sequence identityof at least some amino acids in an amino acid sequence of theinactive-form enzyme specified in step (1) and specifying an amino acidwith relatively low conservation.

In this step, other proteins having high amino acid sequence identitywith the inactive-form enzyme specified in step (1) are used, theidentity is searched by, for example, BLAST(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome)and the respective sequence conservation is calculated on INTMSAlign(NPL 7). Other proteins having high amino acid sequence identity withthe inactive-form enzyme are selected, for example, as follows. Thesequence of an inactive-form enzyme is entered in the section “Sequence”on the BLAST site, 5000 is selected as the value of “Max targetsequences” of the “General Parameters” under the “Algorithm parameters”section below and 1.0E-3 is entered as the value of “Expect threshold”and then the BLAST search is started, thereby other proteins having highidentity can be selected. By downloading all the obtained proteinsequences having high identity in FASTA format and entering thesequences to INTMSAlign, sequence conservation may be calculated.

The sequence conservation may be calculated for the entire amino acidsequence or a part of the amino acid sequence of the inactive-formenzyme. When the sequence conservation is calculated for a part of theamino acid sequence, the part of the sequence may be, for example, anα-helix sequence of the enzyme protein; however, it should be noted thatit is not intended to be limited to an α-helix sequence. The sequencemay be, other than an α-helix sequence, a β-structure, a loop and thelike. However, it should also be noted that it is not intended to belimited to these sequences.

Step (3b)

Step (3b) is the step of preparing a gene having a base sequence thatcodes for an amino acid sequence in which at least one amino acid withrelatively low conservation specified in step (2b) is substituted to anamino acid with higher conservation than said amino acid. In step (3b),preparation of a gene having a base sequence that codes for a mutantamino acid sequence, namely, the procedures other than selection of theamino acid to be substituted which is carried out by the method of step(2b), may be carried out by referring to step (3a) in the producingmethod of the first embodiment of an active-form mutant enzyme of thepresent invention.

Step (4b)

Step (4b) is the step of expressing, in a heterologous expressionsystem, the gene having the base sequence prepared in step (3b) toobtain a protein and selecting from the obtained proteins, a protein(active-form mutant enzyme) that has the same sort of activity as thatobtained in the native form. Step (4b) may be carried out by referringto step (4a) in the producing method of the first embodiment of anactive-form mutant enzyme of the present invention.

Example 3 (expression of luciferase derived from Metridia pacifica as anactive-form mutant enzyme) and Example 4 (expression of amino aciddegradation enzymes derived from Drosophila melanogaster as active-formmutant enzymes focusing on α-helices and amino acid residueconservation) below are examples of the producing method of the secondembodiment.

In Example 3, the amino acid conservation of two amino acid sequenceregions, L76 to A89 (see Table 4) and S176 to I201 (see Table 5), wasrespectively calculated and it was found that 180 and A177 were aminoacids with low conservation. Further, for 180, a mutagenized targetprotein was prepared in which the amino acid was substituted to A havinghigh conservation and for A177, a mutagenized target protein wasprepared in which the amino acid was substituted to D having highconservation. As a result, although the native form was an inactive-formenzyme, active-form mutant enzymes that exhibited activity were obtained(see FIG. 12).

In Example 4, the amino acid conservation of two amino acid sequenceregions, S111 to E118 and A171 to S180 (see Table 6), of ornithinedecarboxylase derived from Drosophila melanogaster (DmODC) wasrespectively calculated and it was found that K117 and L176 were aminoacids having low conservation. For K117, a mutagenized target proteinwas prepared in which the amino acid was substituted to L having highconservation and for L176, a mutagenized target protein was prepared inwhich the amino acid was substituted to E having high conservation. As aresult, although the native form was an inactive-form enzyme,active-form mutant enzymes that exhibited activity were obtained.Similarly, the amino acid conservation of two amino acid sequenceregions, V174 to L189 and G252 to F262 (see Table 7), of glutamatedehydrogenase (DmGluDH) was respectively calculated and it was foundthat V174, K257 and L261 were amino acids having low conservation. ForV174, a mutagenized target protein was prepared in which the amino acidwas substituted to D having high conservation, for K257, a mutagenizedtarget protein was prepared in which the amino acid was substituted to Yhaving high conservation and for L261, a mutagenized target protein wasprepared in which the amino acid was substituted to E having highconservation. As a result, although the native form was an inactive-formenzyme, active-form mutant enzymes that exhibited activity wereobtained.

<Combination of the Method of the First Embodiment and the Method of theSecond Embodiment for Producing an Active-Form Mutant Enzyme of thePresent Invention>

When a protein that forms an enzyme has in the conformation thereoffacing hydrophilic amino acid residues, amino acids on a helix may alsobe hydrophilic amino acids and residues involved in an enzyme reactionin a substrate pocket may be hydrophilic amino acids, and it isparticularly highly possible that amino acid residues having a highsequence conservation are such amino acids. In contrast, amino acidshaving low sequence conservation may be present only in the enzyme andit is presumed that by removing such amino acids, an enzyme activity ofthe protein may be increased. The producing method of the secondembodiment of the present invention utilizes the feature and increasesan enzyme activity by picking up an amino acid with low sequenceconservation and substituting to an amino acid with high sequenceconservation. It is believed that according to the above, a highlyrational mutation can be introduced in order to obtain an active-formmutant enzyme.

The schematic illustration of the above relationship is shown in FIG. 3.Among amino acids that form a protein (the entire circle), there are acircle (left) of amino acids that form an α-helix sequence and a circle(right) of amino acids with low sequence conservation. In the circle(left) of amino acids that form an α-helix sequence, there is a circle(the circle on left) of hydrophobic amino acids in the hydrophilicdomain or hydrophilic amino acids in the hydrophobic domain specified inthe producing method of the first embodiment of the present invention. Acircle (right) of amino acids with low sequence conservation specifiedin the producing method of the second embodiment of the presentinvention may overlap with the circle (left) of amino acids that form anα-helix sequence and the circle (the circle on left) of hydrophobicamino acids in the hydrophilic domain or hydrophilic amino acids in thehydrophobic domain. The overlap may not happen for some proteins.

When the overlap happens, for the inactive-form enzyme specified in step(1), a hydrophobic amino acid that is present in a hydrophilic domainand/or a hydrophilic amino acid that is present in a hydrophobic domainis specified in step (2a) in the method of the first embodiment forproducing an active-form mutant enzyme of the present invention and anamino acid with relatively low conservation is specified in step (2b) inthe method of the second embodiment for producing an active-form mutantenzyme of the present invention. Based on both information, steps (3a)and (4a) (step (3b) and (4b)) are carried out, so that the gene may beexpressed in a heterologous expression system to obtain a protein andfrom the obtained proteins, a protein (active-form mutant enzyme) thatexhibits the same sort of activity as that obtained in the native formmay be selected. In Example 2 (expression of arginine decarboxylasederived from Arabidopsis thaliana as an active-form mutant enzyme), theproducing method of the first embodiment and the method of the secondembodiment for producing an active-form mutant enzyme of the presentinvention are combined.

<Active-Form Mutant Enzyme>

The present invention encompasses a novel active-form mutant enzymeselected by the method of the present invention.

The active-form mutant mandelonitrile oxidase of the present inventionhas an amino acid sequence shown in SEQ ID NO: 2, wherein:

valine at the position 444 has been substituted to threonine, serine,tyrosine, histidine, glutamic acid, glutamine, aspartic acid,asparagine, lysine or arginine and/orvaline at the position 455 has been substituted to glutamic acid,glutamine, aspartic acid, asparagine, lysine or arginine. Theactive-form mutant mandelonitrile oxidase of the present inventionencompasses an enzyme with an activity that is similar to or above theenzyme activity of the native form.

Specifically, the active-form mutant mandelonitrile oxidases of thepresent invention in which valine at the position 455 has beensubstituted to other amino acids respectively exhibit, as shown in FIG.4, mandelonitrile oxidase activity, while the native form has the enzymeactivity that is substantially zero. The active-form mutantmandelonitrile oxidases of the present invention in which valine at theposition 444 has been substituted to other amino acids also respectivelyexhibit, as shown in FIG. 5, mandelonitrile oxidase activity, while thenative form has the enzyme activity that is substantially zero.

The active-form mutant arginine decarboxylase of the present inventionhas an amino acid sequence shown in SEQ ID NO: 3, wherein:

valine at the position 261 has been substituted to threonine, serine,glutamic acid, aspartic acid, asparagine, lysine or arginine and/orarginine at the position 430 has been substituted to valine, leucine oralanine, and leucine at the position 435 has been substituted tohistidine, glutamic acid, glutamine, aspartic acid, asparagine orlysine. The active-form mutant arginine decarboxylase of the presentinvention encompasses an enzyme which is an active-form mutant enzymeand has an activity that is similar to or above the enzyme activity ofthe native form.

The active-form mutant arginine decarboxylases of the present inventionin which valine at the position 261 has been substituted to other aminoacids respectively have, as shown in FIG. 8, arginine decarboxylaseactivity, while the native form has the enzyme activity that issubstantially zero.

The active-form mutant arginine decarboxylases of the present inventionin which arginine at the position 430 has been substituted to otheramino acids respectively have, as shown in FIG. 9, argininedecarboxylase activity, while the native form has the enzyme activitythat is substantially zero.

The active-form mutant arginine decarboxylases of the present inventionin which leucine at the position 435 has been substituted to other aminoacids respectively have, as shown in FIG. 10, arginine decarboxylaseactivity, while the native form has the enzyme activity that issubstantially zero.

The active-form mutant ornithine decarboxylase of the present inventionhas an amino acid sequence shown in SEQ ID NO: 7, wherein:

lysine at the position 117 has been substituted to leucine and/orleucine at the position 176 has been substituted to glutamic acid. Theactive-form mutant ornithine decarboxylase of the present inventionencompasses an enzyme which is an active-form mutant enzyme and has anactivity that is similar to or above the enzyme activity of the nativeform.

Specifically, although the enzyme activity of the native form could notbe observed, the active-form mutant ornithine decarboxylases of thepresent invention in which lysine at the position 117 has beensubstituted to leucine and in which leucine at the position 176 has beensubstituted to glutamic acid in ornithine decarboxylase (DmODC)respectively had activity of 0.45 U/mL and 0.04 U/mL.

The active-form mutant luciferase of the present invention has an aminoacid sequence shown in SEQ ID NO: 5, wherein:

isoleucine at the position 80 has been substituted to lysine and/oralanine at the position 177 has been substituted to aspartic acid. Theactive-form mutant luciferase of the present invention encompasses anenzyme which is an active-form mutant enzyme and has an activity that issimilar to or above the enzyme activity of the native form.

Specifically, the active-form mutant luciferase of the present inventionin which isoleucine at the position 80 has been substituted to lysineand the active-form mutant luciferase (MpLUC) of the present inventionin which alanine at the position 177 has been substituted to asparticacid respectively have, as shown in FIG. 12, luciferase activity, whilethe native form has the enzyme activity that is substantially zero.

The active-form mutant glutamate dehydrogenase of the present inventionhas an amino acid sequence shown in SEQ ID NO: 9, wherein:

valine at the position 174 has been substituted to aspartic acid,lysine at the position 257 has been substituted to tyrosine and/orleucine at the position 261 has been substituted to glutamic acid. Theactive-form mutant glutamate dehydrogenase of the present inventionencompasses an enzyme which is an active-form mutant enzyme and has anactivity that is similar to or above the enzyme activity of the nativeform.

Specifically, the active-form mutant glutamate dehydrogenases of thepresent invention in which valine at the position 174 has beensubstituted to aspartic acid, leucine at the position 257 has beensubstituted to tyrosine and leucine at the position 261 has beensubstituted to glutamic acid in glutamate dehydrogenase (DmGluDH)respectively had activity of 0.14 U/mL, 0.91 U/mL and 0.05 U/mL.

The present invention encompasses a method for producing an active-formmutant enzyme comprising expressing a gene that codes for the amino acidsequence of the active-form mutant enzyme of the present invention in anE. coli expression system to obtain a protein.

The active-form mutant enzyme is, for example, an active-form mutantmandelonitrile oxidase, an active-form mutant arginine decarboxylase, anactive-form mutant ornithine decarboxylase, an active-form mutantluciferase or an active-form mutant glutamate dehydrogenase.

<Method for Producing a Soluble Mutant Protein (Producing Method of theThird Embodiment)>

The method for producing a soluble mutant protein of the presentinvention (producing method of the third embodiment) includes:

expressing, in a heterologous expression system, a gene having a basesequence that codes for an amino acid sequence in which:at least one hydrophobic amino acid that is present in a hydrophilicdomain of an α-helix structure region is substituted (to an amino acidwith higher hydrophilicity or lower hydrophobicity than the amino acidto be substituted) and/or at least one hydrophilic amino acid that ispresent in a hydrophobic domain of the α-helix structure region issubstituted (to an amino acid with higher hydrophobicity or lowerhydrophilicity than the amino acid to be substituted),wherein the α-helix structure region is in an amino acid sequence of aprotein of which native form is a soluble protein but which becomesinsoluble when expressing a gene of the protein in a heterologousexpression system (hereinafter “an insoluble-form protein); andselecting a protein (hereinafter “a soluble mutant protein”) that issoluble.

The method for producing a soluble mutant protein of the presentinvention (producing method of the third embodiment) specificallyincludes the following steps:

(1) specifying a protein of which native form is a soluble protein butwhich becomes insoluble when expressing a gene of the protein in aheterologous expression system (hereinafter “an insoluble-formprotein”);(2a) specifying an α-helix structure region of the insoluble-formprotein specified in step (1), specifying a hydrophilic domain and/orhydrophobic domain of the specified α-helix structure region, andspecifying a hydrophobic amino acid that is present in the hydrophilicdomain and/or a hydrophilic amino acid that is present in thehydrophobic domain;(3a) preparing a gene having a base sequence that codes for an aminoacid sequence in which at least one hydrophobic amino acid that ispresent in the hydrophilic domain of the α-helix structure region in theamino acid sequence of the insoluble-form protein is substituted (to anamino acid with higher hydrophilicity or lower hydrophobicity than theamino acid to be substituted) and/or at least one hydrophilic amino acidthat is present in the hydrophobic domain of the α-helix structureregion is substituted (to an amino acid with higher hydrophobicity orlower hydrophilicity than the amino acid to be substituted); and(4a) expressing, in a heterologous expression system, the gene havingthe base sequence prepared in step (3a) to obtain a protein andselecting from the obtained proteins, a protein (hereinafter “a solublemutant protein”) that is soluble.

The method for producing a soluble mutant protein of the presentinvention (producing method of the third embodiment) may be carried outby substituting the inactive-form enzyme to the insoluble-form proteinand the active-form mutant enzyme to the soluble mutant protein in themethod for producing an active-form mutant enzyme of the presentinvention (producing method of the first embodiment).

<Method for Producing a Soluble Mutant Protein (Producing Method of theFourth Embodiment)>

The method for producing a soluble mutant protein of the presentinvention (producing method of the fourth embodiment) includesexpressing, in a heterologous expression system, a gene having a basesequence that codes for an amino acid sequence of a protein of whichnative form is a soluble protein but which becomes insoluble whenexpressing a gene of the protein in a heterologous expression system(“an insoluble-form protein”), wherein in the amino acid sequence, atleast one amino acid with relatively low conservation is substituted toan amino acid with higher conservation than said amino acid; andobtaining a protein (“a soluble mutant protein”) that is soluble.

The method for producing a soluble mutant protein of the presentinvention (producing method of the fourth embodiment) specificallyincludes the following steps:

(1) specifying a protein of which native form is a soluble protein butwhich becomes insoluble when expressing a gene of the protein in aheterologous expression system (“an insoluble-form protein”);(2b) determining a conservation in sequence identity of at least someamino acids in an amino acid sequence of the insoluble-form proteinspecified in step (1) and specifying an amino acid with relatively lowconservation;(3b) preparing a gene having a base sequence that codes for an aminoacid sequence in which at least one amino acid with relatively lowconservation is substituted to an amino acid with higher conservationthan said amino acid; and(4b) expressing, in a heterologous expression system, the gene havingthe base sequence prepared in step (3b) to obtain a protein andselecting from the obtained proteins, a soluble mutant protein.

The method for producing a soluble mutant protein of the presentinvention (producing method of the fourth embodiment) may be carried outby substituting the inactive-form enzyme to the insoluble-form proteinand the active-form mutant enzyme to the soluble mutant protein in themethod for producing an active-form mutant enzyme of the presentinvention (producing method of the second embodiment).

Similar to the method of the first embodiment and the method of thesecond embodiment for producing an active-form mutant enzyme of thepresent invention, the method for producing a soluble mutant protein ofthe present invention (producing method of the third embodiment) and themethod for producing a soluble mutant protein of the present invention(producing method of the fourth embodiment) may be combined.

In the method for producing a soluble mutant protein of the presentinvention (producing methods of the third and fourth embodiments), beinga soluble mutant protein may be judged from, for example, a solubleprotein amount in an extract after heterologous expression. Further, thesoluble mutant protein may be defined as a mutant protein of which thesoluble protein amount in the extract after heterologous expression ishigher than the soluble protein amount in an extract after heterologousexpression of the native form protein. The soluble protein amount meansthe amount of the soluble protein excluding other soluble proteins thatare present in the extract. The extract after heterologous expressionmay be, as a solution, a buffered aqueous solution without anysurfactant and extraction may be carried out by physically ormechanically disrupting (for example, ultrasonication, grinding, Frenchpress) the host (cells) used for heterologous expression in the presenceof the buffered aqueous solution. The extract may contain, in additionto the soluble mutant protein, an insoluble protein and in this case,the extract is centrifuged to obtain the soluble mutant protein in asupernatant. The soluble mutant protein in a supernatant may be assayedby a well-known protein assay, examples of which may includeelectrophoresis, ELISA, western blotting and the like.

The insoluble-form protein in the present invention is a protein ofwhich native form is a soluble protein but which becomes insoluble in aheterologous expression system when expressing a gene of the protein inthe heterologous expression system. As examples of the heterologousexpression system, the description in the method for producing anactive-form mutant enzyme of the present invention (producing method ofthe first embodiment) may be referred to. Examples of the insoluble-formprotein may include one protein selected from the group consisting of anenzyme, a cytokine, a haemoglobin and a myoglobin. Although not allcytokines, haemoglobins and myoglobins are insoluble-form proteins inthe present invention, some of them are insoluble-form proteins in thepresent invention. As the insoluble-form protein in the presentinvention, one protein selected from the group consisting of IFN-γ,IL-2, IFNβ and human growth hormone may be more specifically mentioned.By referring to the following references, it is found that some of theproteins are insoluble-form proteins of the present invention. Humangrowth hormone (Fibroblast growth factor 15): PLoS ONE, 6, e20307(2011), Interleukin-6 (IL-6): J. Vet. Med. Sci., 66, pp. 1053-1057,(2004)

The present invention is hereinafter described in further detail by wayof Examples. However, Examples merely exemplify the present inventionand it is not intended to limit the present invention to Examples.

Example 1

Expression of Mandelonitrile Oxidase Derived from Chamberliniushualinensis (ChMOX) as an Active-Form Mutant Enzyme

(1) Mutagenesis of a Mandelonitrile Oxidase Gene

A mandelonitrile oxidase gene derived from Chamberlinius hualinensis(SEQ ID NO: 1) was introduced to an E. coli expression vector pET22b toprepare “pET22b-ChMOX”. An aqueous solution of pColdI-ChMOX (5 pt) wasintroduced into E. coli XL-1 Red (manufactured by Agilent Technologies)by heat shock for transformation. The cells were cultured in LB mediumcontaining 100 μg/mL of ampicillin for 48 hours, and the colonies thusgrown were suspended in a 100 mM potassium phosphate buffer (pH 7.0) byusing a bacteria spreader to collect the cells. The plasmid vector wasextracted to obtain a mutant enzyme library.

(2) Selection of an Active-Form Mutant Enzyme Expression Plasmid Vectorfrom the Mutant Mandelonitrile Oxidase Library

The mutant enzyme library obtained in (1) was introduced into E. coliBL21 (DE3) by heat shock under the same conditions as in (1) fortransformation. Colonies grown on LB agar medium containing 100 μg/mL ofampicillin were cultured in wells of a 96-well deep well platecontaining 300 μL of LB medium containing 100 μg/mL of ampicillin at 37°C., 0.5 μg/mL of IPTG was added at a cell turbidity of about 0.5 and thecells were cultured at 30° C. for 6 hours. After collecting the cells bycentrifugation (2000×g, 15 minutes, 4° C.), 50 μL of a cell lysisreagent BugBuster (manufactured by Novagen) was added and the mixturewas shaken at room temperature for about 15 minutes. A 10 mM potassiumphosphate buffer (pH 7.0, 150 μL) was then added and a supernatant wasobtained by centrifugation (2000×g, 15 minutes, 4° C.) to give a crudeenzyme solution. By referring to PTL 10, the crude enzyme solution wasmeasured for mandelonitrile oxidase activity. The mutant enzyme whichexhibited the activity was sequenced for the DNA sequence on a DNAsequencer. As a result, it was found that valine at the position 455 wassubstituted to alanine.

(3) Saturation Mutation of the Amino Acid at the Position 455 inMandelonitrile Oxidase

Mutant mandelonitrile oxidase expression plasmids were constructed inwhich valine at the position 455 was substituted to other 19 aminoacids. Each plasmid was cultured under the same conditions as in (1) andthe mandelonitrile oxidase activity was assayed. As a result, it wasfound that, as shown in FIG. 4, the activity was high when thesubstituting amino acid had lower hydropathy index, namely higherhydrophilicity than valine and the mandelonitrile oxidase activity wasparticularly high for hydrophilic amino acids (E, Q, D, N, K and R).

(4) Analysis Using Secondary Structure Prediction and Helical Wheel

As a result of prediction on a secondary structure predicting programmePSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/) of the amino acid sequenceof mandelonitrile oxidase (SEQ ID NO: 2), it was revealed that valine atthe mutation site, the position 455, was present in the amino acidsequence (RVDIDTMVRGVHVALNFG) of an α-helix structure (FIG. 1A). Inaddition, as a result of drawing a helical wheel on pepwheel(http://emboss.bioinformatics.nl/cgi-bin/emboss/pepwheel), valine at theposition 455 was, as shown in FIG. 1B, a hydrophobic amino acid that waspresent in a hydrophilic domain. It was revealed that substitution ofvaline at the position 455 to alanine was introduction of a mutation inan α-helix and further a mutation to an amino acid with low hydropathyindex, namely high hydrophilicity that was present in a hydrophilicdomain.

(5) Introduction of Saturation Mutation of Valine at the Position 444

As, similar to valine at the position 455, V444, G452 and F459 are also“hydrophobic amino acids that are present in a hydrophilic domain” of anα-helix, mutations were introduced at V444, G452 and F459 by usingQuikChange Site-Directed Mutagenesis Kit (manufactured by AgilentTechnologies) so that the sites were substituted to other 19 aminoacids. E. coli JM109 (DE3) was transformed with each of the obtainedmutagenized plasmids, inoculated to LB plates containing ampicillin andcultured at 37° C. for 16 hours. The plasmids harboured by the obtainedcolonies were sequenced and mutant mandelonitrile oxidase expressionplasmids were constructed in which V444, G452 and F459 were substitutedto other 19 amino acids. Each plasmid was cultured under the sameconditions as in (1) and the mandelonitrile oxidase activity wasassayed. It was revealed that mutations at V444 to amino acids with lowhydropathy index, namely high hydrophilicity were correlated toexpression of active-form enzymes. Activity was not exhibited by aminoacid substitutions to other 19 amino acids at G452 or F459. From theresult shown in FIGS. 6A-6C obtained by re-drawing a helical wheelaccording to the drawing manner shown in FIGS. 2A-2B, it was found thatthe two residues are present at the border between a hydrophilic domainand a hydrophobic domain as shown in FIG. 6C, and thus it was believedthat the drawing manner shown in FIGS. 2A-2B was suitable for dividingthe regions.

(6) Sequence Conservation at V444, G452, V455 and F459

Homology was searched on BLAST(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome)and the sequence conservation of the α-helix sequence was calculated onINTMSAlign (NPL 7). The sequence conservation was calculated under thefollowing conditions. The sequence of the inactive-form enzyme isentered in the section “Sequence” on the BLAST site, 5000 is selected asthe value of “Max target sequences” of the “General Parameters” underthe “Algorithm parameters” section below and 1.0E-3 is entered as thevalue of “Expect threshold” and then the BLAST search is started,thereby other proteins with high identity are selected. By downloadingall the obtained protein sequences having high identity in FASTA formatand entering the sequences to INTMSAlign, sequence conservation wascalculated.

The results of conservation of V444, G452, V455 and F459 are shown inTable 2. It was found that at V444 and V455, relatively, not onlyhydrophobic amino acids but also hydrophilic amino acids were frequentlyconserved, while at G452 and F459, amino acids with similar hydropathyindex were conserved.

According to the above, it was suggested that when, particularly, ahydrophilic amino acid is conserved at a hydrophobic amino acid in ahydrophilic domain, expression of an active-form mutant enzyme isfacilitated by substituting the hydrophobic amino acid to a hydrophilicamino acid.

TABLE 2 Conservation of amino acids at V444, G452, V455 and F459 ResidueI V L F C M A G T W S Y P H E Q D N K R Non V444 1.4 0.7 3.1 1.7 0.0 0.64.3 0.5 2.9 0.2 1.8 4.2 0.1 2.3 8.8 4.5 6.9 0.8 2.3 5.8 47.0 G452 0.20.5 0.2 0.1 18.3 0.5 17.1 57.2 0.5 0.0 4.0 0.2 0.0 0.1 0.0 0.1 0.0 0.00.3 0.1 0.6 V455 17.8 3.3 41.5 7.0 0.3 6.4 5.2 0.0 2.4 0.9 0.4 1.4 0.11.1 1.9 2.5 0.1 0.1 4.6 2.6 0.2 F459 64.4 7.5 21.8 0.7 0.0 3.2 0.4 0.00.2 0.1 0.1 0.1 0.0 0.0 0.1 0.1 0.1 0.1 0.1 0.1 1.0

Example 2

Expression of Arginine Decarboxylase Derived from Arabidopsis thaliana(AtADC) as an Active-Form Mutant Enzyme

(1) Estimation of α-Helices by a Secondary Structure PredictingProgramme and Drawing of a Helical Wheel

The amino acid sequence of arginine decarboxylase derived fromArabidopsis thaliana (SEQ ID NO: 3) was analysed with the secondarystructure predicting programme PSIPRED as described above and, as aresult, it was estimated that the enzyme had 20 α-helices (FIG. 7A). Bydrawing helical wheels by using all sequences of the α-helices asdescribed above, it was revealed that valine at the position 261,leucine at the position of 264, arginine at the position of 430, leucineat the position of 435 and lysine at the position of 441 in the 8th(TVQILRWRKLSQ) and 13th (RESCLLYVDQLKQRCVE) α-helices from theN-terminal were, as shown in FIGS. 7B and 7C, “a hydrophobic amino acidthat is present in a hydrophilic domain” or “a hydrophilic amino acidthat is present in a hydrophobic domain”.

(2) Mutagenesis at V261, L264, R430, L435 and K441 of ArginineDecarboxylase

An arginine decarboxylase gene derived from Arabidopsis thaliana (SEQ IDNO: 4) was introduced to pET11a to prepare “pET11a-AtArgDC”. Mutationswere introduced by using QuikChange Site-Directed Mutagenesis Kit(manufactured by Agilent Technologies) so that the residues described in(1) were substituted to other amino acids. E. coli JM109 (DE3) wastransformed with each of the obtained mutagenized plasmids, inoculatedto LB plates containing ampicillin and cultured at 37° C. for 16 hours.The plasmids harboured by the obtained colonies were sequenced andmutant arginine decarboxylase expression plasmids were constructed inwhich V261, L264, R430, L435 and K441 were substituted to other 19 aminoacids. Each plasmid was cultured under the same conditions as above andthe arginine decarboxylase activity was assayed. It was revealed thatmutations at V261 (FIG. 8) and L435 (FIG. 9) to amino acids with lowhydropathy index, namely high hydrophilicity and at R430 (FIG. 10) toamino acids with high hydropathy index, namely high hydrophobicity werecorrelated to expression of active-form mutant enzymes. Introduction ofcombined mutations of the above increased the activity, and thus anincrease of the production of active-form mutant enzymes by accumulationof mutations was observed. However, activity was not exhibited by aminoacid substitutions to other 19 amino acids at L264 or K441.

(3) Sequence Conservation at V261, L264, R430, L435 and K441

Homology was searched on BLAST and the sequence conservation of theα-helix sequences was calculated on INTMSAlign (NPL 7). The sequenceconservation was calculated under the following conditions. The sequenceof the inactive-form enzyme is entered in the section “Sequence” on theBLAST site, 5000 is selected as the value of “Max target sequences” ofthe “General Parameters” under the “Algorithm parameters” section belowand 1.0E-3 is entered as the value of “Expect threshold” and then theBLAST search is started, thereby other proteins with high identity areselected. By downloading all the obtained protein sequences having highidentity in FASTA format and entering the sequences to INTMSAlign,sequence conservation was calculated.

The results of conservation of V261, L264, R430, L435 and K441 are shownin Table 3. It was found that at V261 and L435, relatively, not onlyhydrophobic amino acids but also hydrophilic amino acids were frequentlyconserved and at R430, relatively, not only hydrophilic amino acids butalso hydrophobic amino acids were frequently conserved. However, at L264and K441, it was found that amino acids with similar hydropathy indexwere conserved.

According to the above, it was suggested that an amino acid particularlyhaving the same characteristics as the region is conserved when at ahydrophobic amino acid in a hydrophilic domain or a hydrophilic aminoacid in a hydrophobic domain, expression of an active-form mutant enzymeis facilitated by substituting such an amino acid.

TABLE 3 Conservation of amino acids at V261, L264, R430, L435 and K441Residue I V L F C M A G T W S Y P H E Q D N K R Non V261 4.4 0.3 2.4 1.70.0 0.8 13.3 1.3 21.6 0.5 16.4 0.9 5.4 0.9 5.6 0.7 5.8 3.8 6.7 3.4 4.0L264 8.2 10.1 49.9 0.6 0.0 2.8 12.9 1.5 0.3 0.0 0.5 1.0 1.1 0.1 1.7 1.10.7 1.0 0.3 0.6 5.5 R430 2.5 5.0 23.8 1.5 0.5 3.8 9.2 1.1 0.6 0.7 1.26.8 1.8 1.3 1.5 1.7 0.9 1.0 0.9 1.2 33.1 L435 1.4 1.4 4.5 0.7 0.3 1.31.1 0.6 1.2 3.4 1.0 2.0 0.9 30.7 1.7 2.9 1.8 9.3 0.6 0.9 32.3 K441 2.92.6 21.0 1.8 0.5 5.1 1.5 1.4 3.4 0.4 1.0 0.5 0.8 3.3 0.9 1.2 1.0 0.810.8 8.1 31.1

Example 3

Expression of Luciferase Derived from Metridia pacifica (MpLUC) as anActive-Form Mutant Enzyme

(1) Estimation of α-Helices by a Secondary Structure PredictingProgramme

As a result of prediction of a secondary structure of the amino acidsequence (SEQ ID NO: 5) of luciferase (MpLUC) 1-1 derived from marineplankton, Metridia pacifica, with the same programme as above (FIGS.11A-11B), it was found that as shown in FIG. 11A, MpLUC 1-1 had 6α-helix structures and the α-helix sequence indicated with (1) could bedivided into the hydrophilic domain and the hydrophobic domain based onthe helical wheel prepared (FIG. 11B). However, the α-helix sequenceindicated with (2) could not be divided into a hydrophilic domain and ahydrophobic domain based on the helical wheel prepared. For α-helixsequences (1) and (2), homology was searched on BLAST and the sequenceconservation of the α-helix sequences was calculated on INTMSAlign (NPL7). The sequence conservation was calculated under the followingconditions. The sequence of the inactive-form enzyme is entered in thesection “Sequence” on the BLAST site, 5000 is selected as the value of“Max target sequences” of the “General Parameters” under the “Algorithmparameters” section below and 1.0E-3 is entered as the value of “Expectthreshold” and then the BLAST search is started, thereby other proteinswith high identity are selected. By downloading all the obtained proteinsequences having high identity in FASTA format and entering thesequences to INTMSAlign, sequence conservation was calculated.

As a result, isoleucine at the position 80 and alanine at the position177 were found to be amino acids with low conservation and it was alsorevealed that lysine and aspartic acid were conserved with highpercentages at the former and latter amino acids, respectively.

TABLE 4 Sequence conservation at L76 to A89 of MpLUC Residue I V L F C MA G T W S Y P H E Q D N K R Non L76 5.4 2.7 59.2 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 8.3 0.0 0.0 0.0 0.0 0.0 22.1 0.0 2.3 E77 0.0 0.0 0.0 0.0 0.00.0 27.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 62.0 0.0 8.3 0.0 0.0 0.0 2.2 V782.7 86.5 8.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 2.2 L79 2.7 0.0 92.7 0.0 0.0 2.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 2.2 I80 14.6 5.3 3.1 0.0 0.0 5.1 11.3 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 6.1 0.0 50.2 2.2 2.1 E81 12.9 0.0 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 84.9 0.0 0.0 0.0 0.0 0.0 2.2 M820.0 0.0 2.2 0.0 0.0 95.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 2.2 E83 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 8.689.3 0.0 0.0 0.0 0.0 0.0 2.1 A84 0.0 0.0 0.0 0.0 6.1 0.0 91.7 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.2 N85 8.6 0.0 0.0 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 89.3 0.0 0.0 2.1 A86 0.00.0 0.0 0.0 0.0 0.0 97.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 2.2 R87 0.0 11.3 0.0 10.6 0.0 0.0 3.1 0.0 0.0 0.0 5.8 0.0 0.0 0.00.0 5.5 0.0 0.0 20.0 41.4 2.3 K88 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 0.0 0.0 2.4 0.0 0.0 8.6 51.5 35.4 2.1 A89 0.0 0.0 0.0 0.00.0 0.0 89.0 0.0 0.0 0.0 8.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.2

TABLE 5 Sequence conservation at S176 to I201 of MpLUC Residue I V L F CM A G T W S S176 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 84.9 A177 0.00.0 0.0 0.0 6.1 0.0 23.4 0.0 0.0 0.0 0.0 L178 0.0 0.0 77.4 0.0 0.0 0.011.3 0.0 0.0 0.0 0.0 L179 0.0 0.0 100.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0K180 0.0 0.0 0.0 5.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 K181 0.0 0.0 0.0 0.00.0 0.0 8.6 0.0 0.0 0.0 0.0 W182 0.0 2.7 0.0 0.0 0.0 0.0 13.8 0.0 0.083.5 0.0 L183 0.0 0.0 97.6 0.0 0.0 2.4 0.0 0.0 0.0 0.0 0.0 P184 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 D185 0.0 0.0 0.0 0.0 0.0 0.0 2.7 2.411.6 0.0 14.2 R186 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 C187 0.00.0 0.0 0.0 100.0 0.0 0.0 0.0 0.0 0.0 0.0 A188 0.0 0.0 0.0 0.0 0.0 0.049.8 0.0 22.7 0.0 8.1 S189 0.0 0.0 0.0 0.0 0.0 0.0 0.0 19.0 41.5 0.028.6 F190 0.0 0.0 0.0 100.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 A191 0.0 0.0 0.00.0 0.0 0.0 86.0 0.0 0.0 0.0 0.0 D192 0.0 2.8 2.7 0.0 0.0 0.0 6.1 0.019.9 0.0 32.9 K193 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 I194 94.20.0 0.0 0.0 0.0 5.8 0.0 0.0 0.0 0.0 0.0 Q195 0.0 0.0 2.4 0.0 0.0 0.0 0.00.0 0.0 0.0 0.0 S196 0.0 0.0 0.0 0.0 0.0 0.0 2.7 27.3 0.0 0.0 34.1 E1970.0 0.0 0.0 0.0 0.0 0.0 2.9 0.0 0.0 0.0 0.0 V198 2.8 84.6 0.0 0.0 0.03.1 6.1 3.5 0.0 0.0 0.0 D199 0.0 0.0 0.0 0.0 0.0 0.0 1.4 2.7 0.0 0.0 3.1N200 0.0 1.7 0.0 0.0 0.0 0.0 0.1 0.0 28.3 0.0 10.1 I201 87.6 6.1 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 Residue Y P H E Q D N K R Non S176 0.0 0.00.0 0.0 0.0 0.0 15.1 0.0 0.0 0.0 A177 0.0 0.0 0.0 11.0 0.0 54.2 0.0 5.30.0 0.0 L178 0.0 0.0 0.0 0.0 5.3 0.0 0.0 6.1 0.0 0.0 L179 0.0 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 K180 6.1 0.0 0.0 0.0 0.0 0.0 0.0 88.7 0.00.0 K181 0.0 0.0 0.0 0.0 0.0 5.3 0.0 86.2 0.0 0.0 W182 0.0 0.0 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 L183 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0P184 0.0 100.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 D185 0.0 0.0 0.0 0.0 35.827.4 0.0 5.8 0.0 0.0 R186 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 100.0 0.0 C1870.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 A188 0.0 0.0 0.0 0.0 0.0 0.0 0.016.6 2.8 0.0 S189 0.0 0.0 0.0 0.0 8.1 0.0 2.8 0.0 0.0 0.0 F190 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 A191 0.0 0.0 0.0 0.0 6.1 0.0 0.0 2.7 5.30.0 D192 3.1 0.0 0.0 0.0 0.0 27.0 5.5 0.0 0.0 0.0 K193 0.0 0.0 0.0 0.08.0 0.0 8.6 83.5 0.0 0.0 I194 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0Q195 0.0 0.0 0.0 0.0 91.5 0.0 0.0 6.1 0.0 0.0 S196 0.0 0.0 0.0 0.0 0.00.0 4.8 28.5 2.7 0.0 E197 0.0 0.0 0.0 58.3 36.5 0.0 0.0 0.0 2.2 0.0 V1980.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 D199 0.0 0.0 24.3 3.0 0.0 63.80.0 0.0 0.0 1.7 N200 0.0 0.0 0.0 0.0 0.0 0.0 28.5 29.8 0.0 1.5 I201 0.00.0 0.0 0.0 0.0 1.7 3.1 0.0 0.0 1.5

(2) Substitution of “a Hydrophobic Amino Acid that is Present in aHydrophilic Domain of an α-Helix Structure or a Hydrophilic Amino Acidthat is Present in a Hydrophobic Domain of the Region” to a Residue withHigh Conservation

Mutations were introduced in the same manner as in Example 2 (2) so thatthe above amino acids were respectively substituted to other aminoacids. E. coli JM109 (DE3) was transformed with each of the obtainedmutagenized plasmids, inoculated to LB plates containing ampicillin andcultured at 37° C. for 16 hours. The obtained colonies were cultured in5 mL of LB liquid medium containing IPTG, 0.5 μg/mL of IPTG was added ata cell turbidity of about 0.5 and the cells were cultured at 16° C. for12 hours. After collecting the cells by centrifugation (10,000×g, 2minutes, 4° C.), the cells were resuspended in 250 pt of 20 mM potassiumphosphate buffer (pH 7.0) and a crude enzyme solution was prepared byultrasonication and centrifugation. Coelenterazine was added to theobtained crude enzyme solution and luminescence was detected on aluminometer. As a result, luminescence was detected for the group ofenzymes to which mutations were introduced as shown in FIG. 12. Anincrease of luminescence was also detected by introducing A177D mutationto the 180K mutant enzyme. The mutagenized enzyme was purified to asingle protein as shown in FIG. 13 with His GraviTrap (manufactured byGE Healthcare Japan) and it was confirmed that the enzyme was anactive-form mutant enzyme. From the above results, it was revealed thatan active-form mutant enzyme could be expressed by substituting ahydrophobic amino acid that was present in a hydrophilic domain of anα-helix or a hydrophilic amino acid that was present in a hydrophobicdomain of the region to an amino acid with high conservation.

Example 4

Expression of Amino Acid Decomposing Enzymes Derived from Drosophilamelanogaster as Active-Form Mutant Enzymes by Focusing on α-Helices andAmino Acid Residue Conservation

(1) Estimation of α-Helices by a Secondary Structure PredictingProgramme, Drawing of a Helical Wheel and Search of Conserved Amino AcidResidues

The secondary structures of the amino acid sequence (SEQ ID NO: 7) ofornithine decarboxylase (DmODC) and the amino acid sequence (SEQ ID NO:9) of glutamate dehydrogenase (DmGluDH) derived from Drosophilamelanogaster were predicted and α-helix sequences were specified (FIGS.14 and 15). Further, analyses were carried out by focusing on thesequence conservation in the α-helix sequences and as a result, it wasrevealed that, as shown in Tables 6 and 7, the probability was high thatin DmODC, leucine was conserved at the position 117 of lysine andglutamic acid was conserved at the position 176 of leucine, and inDmGluDH, aspartic acid was conserved at the position 174 of valine,tyrosine was conserved at the position 257 of lysine and glutamic acidwas conserved at the position 261 of leucine.

TABLE 6 Sequence conservation at S111 to E118 and A171 to S180 of DmODCResidue I V L F C M A G T W S Y P H E Q D N K R Non S111 0.5 1.1 0.5 4.30.2 0.1 17.5 1.3 1.5 0.8 28.2 0.5 1.0 1.6 8.3 1.6 7.5 1.5 7.0 13.2 1.9H112 3.7 0.2 0.6 3.2 0.4 0.2 3.1 1.1 0.3 0.1 2.8 4.2 0.1 8.4 34.6 10.723.4 0.8 0.1 0.1 1.9 L113 59.3 7.0 14.5 0.4 0.1 10.4 1.3 0.0 0.5 0.0 0.41.4 0.0 0.1 0.5 0.4 0.2 0.0 0.6 1.0 1.9 E114 2.0 1.7 2.3 0.1 0.1 0.218.7 0.9 1.2 0.2 1.1 0.3 0.1 0.7 12.2 3.0 6.7 1.0 18.3 27.4 1.9 Y115 0.41.0 6.0 11.3 0.4 1.3 9.6 0.2 1.5 0.7 0.6 31.6 0.1 5.2 7.7 6.8 1.3 0.50.8 11.3 1.9 A116 0.4 3.2 0.9 6.1 1.0 0.0 79.6 2.4 0.2 0.1 0.6 2.6 0.10.0 0.4 0.0 0.2 0.0 0.1 0.1 2.0 K117 3.2 4.0 30.5 5.3 0.6 0.8 22.5 0.40.3 0.5 2.6 13.3 0.1 6.0 1.3 0.8 0.2 0.5 2.1 3.2 1.9 E118 0.1 0.6 1.00.6 0.1 0.2 13.9 1.8 2.7 0.1 9.1 0.2 0.0 1.1 23.3 7.0 7.6 4.5 18.8 5.52.1 A171 3.4 11.8 9.0 1.1 9.1 1.6 24.9 1.3 3.3 1.0 5.0 0.6 3.9 0.5 4.50.7 0.9 0.8 2.4 11.8 2.4 A172 1.7 8.1 4.1 0.3 0.2 3.9 10.3 2.6 2.1 0.12.7 1.1 6.0 1.3 17.4 3.3 8.0 1.3 4.3 16.5 4.7 A173 4.1 15.6 14.1 0.9 0.46.7 5.8 2.7 2.0 0.1 2.6 0.4 3.4 10.3 7.6 1.9 12.0 1.1 1.5 3.9 3.0 L1744.9 9.0 50.0 6.7 0.2 1.3 1.8 0.7 0.2 0.0 0.1 20.4 0.1 0.0 1.7 0.1 0.40.1 0.2 0.3 1.3 M175 5.0 3.1 55.4 0.6 0.5 2.7 12.3 0.2 0.8 0.0 0.7 0.50.5 0.6 2.9 2.6 1.3 0.6 3.3 5.0 1.1 L176 5.7 4.2 8.5 0.8 0.2 1.1 6.2 1.02.0 4.1 1.9 2.7 0.8 4.3 21.8 7.0 3.3 0.9 6.1 15.9 1.4 L177 3.6 5.4 14.51.1 4.2 1.9 34.5 0.2 4.5 0.2 1.6 3.2 0.1 3.9 2.0 1.9 0.6 2.2 3.2 9.8 1.6A178 1.2 2.5 1.9 0.1 0.6 0.6 68.2 0.9 1.0 0.0 2.0 0.3 0.1 1.7 0.9 1.20.6 0.7 10.0 4.4 1.1 K179 0.5 1.6 0.9 0.4 0.2 0.2 9.1 1.6 3.6 0.0 5.30.4 0.1 5.7 4.5 6.1 3.1 3.5 33.9 17.4 2.2 S180 0.3 0.7 19.5 0.4 0.3 2.48.2 1.5 2.8 0.3 9.3 0.6 0.1 1.6 19.9 5.9 9.5 1.7 5.5 7.0 2.4

TABLE 7 Sequence conservation at V174 to L189 and G252 to L261 ofDmGluDH Residue I V L F C M A G T W S V174 4.6 2.1 3.0 6.9 0.1 2.2 3.10.4 3.2 0.0 2.9 D175 0.1 0.2 1.4 0.5 0.1 1.5 14.8 20.2 2.3 1.0 9.5 E1760.0 0.1 0.0 0.0 0.0 0.1 0.1 0.0 0.2 0.0 0.1 L177 12.0 14.2 63.5 0.0 0.11.7 0.1 0.1 0.8 0.1 0.0 Q178 0.1 0.1 0.3 0.1 0.1 18.5 0.1 0.0 0.0 0.00.0 T179 0.1 0.2 2.1 0.0 0.0 0.2 4.3 1.1 0.0 0.0 1.2 I180 22.2 5.6 49.018.6 0.0 2.3 0.1 0.0 0.0 0.1 0.1 T181 0.7 1.6 0.1 0.2 17.8 0.8 2.9 0.052.4 0.0 21.7 R182 0.0 0.0 0.3 0.0 0.0 0.0 0.1 0.1 0.0 0.0 0.1 R183 0.04.0 0.0 0.0 0.0 0.0 11.0 18.1 0.5 0.0 15.9 Y184 0.3 0.1 0.9 48.8 0.0 0.20.1 0.0 0.0 0.8 0.1 T185 7.6 15.6 0.1 0.9 0.1 20.0 14.7 0.3 38.0 0.0 0.8M186 2.2 1.7 4.5 1.0 0.3 3.7 4.3 0.1 16.5 0.1 20.5 E187 0.2 0.3 0.8 0.00.1 0.5 21.9 2.7 0.2 0.1 1.1 L188 44.0 2.8 43.8 2.9 0.1 3.1 0.2 0.0 0.20.1 0.2 L189 6.7 2.2 0.2 1.5 2.7 0.8 20.0 13.2 0.7 2.0 19.7 G252 0.0 0.00.0 0.0 0.0 0.0 14.8 81.3 0.0 0.0 2.8 R253 0.1 0.1 8.5 5.0 0.1 0.6 0.40.0 1.0 0.3 0.3 G254 0.0 0.2 0.1 0.0 0.0 0.0 0.0 92.1 0.0 0.0 6.3 V2551.7 63.9 12.1 0.0 3.8 0.1 6.8 2.6 3.6 0.0 0.5 W256 3.4 20.7 8.1 26.8 2.35.4 11.4 0.6 7.5 0.0 1.6 K257 19.6 16.2 7.1 9.3 0.1 0.4 0.3 0.0 2.3 0.00.2 A258 5.0 14.7 4.0 12.2 8.4 2.0 15.7 11.2 15.6 0.1 5.0 G259 24.1 9.914.3 0.3 0.8 1.5 17.8 12.5 16.3 0.0 0.6 D260 0.6 2.2 6.2 0.3 2.8 2.0 2.40.5 1.0 0.1 3.5 L261 0.5 1.4 1.6 1.0 0.4 0.1 10.7 0.1 0.9 0.1 2.8 F2623.0 8.6 12.3 11.8 0.6 14.0 40.4 0.4 1.1 1.9 0.4 Residue Y F H E Q D N KR Non V174 0.1 5.1 0.8 15.8 5.0 24.3 3.9 8.7 6.1 1.8 D175 0.8 0.7 6.11.9 2.3 2.7 10.7 2.9 18.6 1.8 E176 0.0 0.0 0.1 95.3 0.8 0.7 0.3 0.0 0.11.8 L177 0.0 0.1 0.1 0.0 0.9 0.0 0.1 3.5 0.9 1.8 Q178 0.1 0.1 0.1 75.71.2 0.0 0.5 0.1 1.2 1.8 T179 0.0 0.0 0.5 0.9 3.0 0.3 1.5 12.0 71.7 1.8I180 0.1 0.0 0.0 0.2 0.0 0.0 0.1 0.1 0.0 1.7 T181 0.0 0.1 0.0 0.0 0.00.0 0.1 0.0 0.1 1.7 R182 0.0 0.0 0.2 0.1 18.3 0.0 0.0 1.1 78.0 1.7 R1830.0 0.0 0.5 0.3 0.5 0.0 0.8 2.8 43.2 2.2 Y184 46.4 0.0 0.0 0.0 0.0 0.00.0 0.1 0.1 2.2 T185 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.1 1.8 M186 4.40.1 1.3 5.5 10.4 2.5 0.8 1.8 16.4 1.9 E187 0.4 0.0 0.2 60.5 1.4 0.2 0.45.1 1.8 2.2 L188 0.1 0.0 0.0 0.0 0.3 0.0 0.1 0.0 0.3 2.0 L189 9.2 0.15.4 1.1 4.1 0.1 2.0 0.6 6.1 1.8 G252 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.01.0 R253 20.4 0.0 0.4 0.3 2.9 0.1 0.0 6.8 51.7 1.0 G254 0.0 0.0 0.0 0.00.0 0.0 0.0 0.0 0.1 1.2 V255 0.0 0.0 0.0 0.0 0.1 0.3 2.8 0.0 0.0 1.6W256 4.6 0.0 0.1 0.1 4.8 0.0 1.3 0.1 0.1 1.5 K257 32.6 0.0 9.9 0.2 0.40.1 0.3 0.0 0.1 1.5 A258 4.0 0.0 0.0 0.0 0.0 0.0 0.4 0.0 0.1 1.5 G2590.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 1.5 D260 0.3 0.0 1.1 24.5 9.7 3.13.0 7.7 27.2 2.0 L261 0.9 0.0 2.4 55.1 5.1 0.7 10.6 1.2 1.9 2.7 F262 2.20.1 0.1 0.1 0.2 0.0 0.0 0.1 0.0 2.5

(2) Introduction of Mutations to the Residues Selected by the Method ofthe Present Invention and Activity Assay

Mutagenesis was made in the same manner as in Example 2 (2) so that eachresidue was substituted to other amino acids. Thereafter, the obtainedcolonies were cultured and a crude enzyme solution was prepared. As aresult, although enzyme activities were not detected for enzymes beforemutagenesis because the enzymes were not expressed as soluble proteins,mutant enzymes of DmODC in which lysine at the position of 117 wassubstituted to leucine and leucine at the position 176 was substitutedto glutamic acid had ornithine decarboxylase activity of 0.45 U/mL and0.04 U/mL, respectively. Mutant enzymes of DmGluDH in which valine atthe position 174 was substituted to aspartic acid, leucine at theposition 257 was substituted to tyrosine and leucine at the position 261was substituted to glutamic acid had glutamate dehydrogenase activity of0.14 U/mL, 0.91 U/mL and 0.05 U/mL, respectively. From the aboveresults, it was revealed that, similar to Example 3, an active-formmutant enzyme could be expressed by substituting a hydrophobic aminoacid that was present in a hydrophilic domain of an α-helix or ahydrophilic amino acid that was present in a hydrophobic domain of theregion to an amino acid with high conservation, even for other enzymes.

Example 5

The procedures in Example 1 (1) to (3) were repeated to examine theexpression level of insoluble proteins and the expression level ofsoluble proteins for the wild type enzyme (WT) and the mutant enzymes(V455D and V455Q) in E. coli. The crude enzyme solution was centrifugedto obtain a supernatant which was a soluble fraction (soluble protein)and a pellet (precipitate) which was an insoluble fraction (insolubleprotein).

In order to examine the expression level of the wild type enzyme (WT)and mutant enzymes (V455D and V455Q) of mandelonitrile oxidase derivedfrom Chamberlinius hualienensis in E. coli, the assay was carried out bywestern blotting using an antibody. After inducing under the sameconditions and preparing crude enzyme solutions (buffer: 10 mM potassiumphosphate buffer (pH 7.0)), the supernatant (soluble fraction) andsupernatant (soluble fraction)+pellet (insoluble fraction) wereelectrophoresed by SDS-PAGE and transferred onto PVDF membranes. Byusing an antibody anti-His tag mAb HRP DirectT (MBL Co., Ltd., Nagoya,Japan) directed to the histidine tag added to proteins, the amount ofthe enzyme in each fraction was determined. As a result, it was revealedthat, although the total expression levels were similar for all enzymes,there was a difference in the expression level in the soluble fractionbetween mutant enzymes (V455D and V455Q) and the wild type enzyme (WT)(FIG. 16).

Example 6

Expression of Human Growth Hormone as a Soluble Protein

It is known that human growth hormone (hGH) is expressed as an insolubleprotein in E. coli. The conformation (PDB ID:3HHR) for hGH has beenpublished and the protein contains 7 α-helix structures. In order toexpress the protein in a soluble form in E. coli, the sequenceconservation of the α-helix sequences was calculated on INTMSAlign (NPL7). The sequence conservation was calculated under the followingconditions. The sequence of the wild type enzyme is entered in thesection “Sequence” on the BLAST site, 5000 is selected as the value of“Max target sequences” of the “General Parameters” under the “Algorithmparameters” section below and 1.0E-3 is entered as the value of “Expectthreshold” and then the BLAST search is started, thereby other proteinswith high identity are selected. By downloading all the obtained proteinsequences having high identity in FASTA format and entering thesequences to INTMSAlign, sequence conservation was calculated.

As a result, leucine at the position 46, phenylalanine at the positionof 55, leucine at the position of 82, leucine at the position of 88,arginine at the position of 95, valine at the position of 97 andisoleucine at the position of 114 were found to be amino acids with lowconservation and it was revealed that lysine, histidine, arginine,glutamic acid, serine, glutamic acid and lysine, respectively, wereconserved at high percentages.

TABLE 8 Sequence conservation at L46, F55, L82, L88, R95, V97 and I114of hGH Residue I V L F C M A G T W S Y P H E Q D N K R Non L46 3.7 2.18.3 10.7 0.2 2.0 3.5 0.4 1.1 0.2 6.5 2.3 0.6 0.6 0.9 9.6 0.2 7.3 12.21.8 25.9 F55 2.3 0.3 1.0 20.2 0.2 0.4 0.6 0.1 0.5 0.1 0.7 11.6 1.3 30.60.2 0.3 0.0 14.9 0.1 0.2 14.5 L82 2.5 10.0 23.8 0.2 0.2 4.2 2.3 6.8 1.70.0 12.1 0.2 0.0 0.7 0.3 0.3 0.2 2.5 3.7 19.8 8.4 L88 3.8 0.9 33.2 1.10.0 0.8 0.6 0.4 0.9 0.1 3.2 0.5 0.4 0.5 19.2 1.4 2.5 16.0 4.8 1.1 8.4R95 2.1 16.9 1.2 0.0 0.1 0.2 5.1 2.8 1.3 0.3 41.1 0.6 0.4 1.3 0.5 3.90.3 3.1 0.3 9.4 9.2 Y97 5.4 28.3 1.8 0.2 0.2 5.0 4.6 4.8 9.6 0.0 4.7 0.70.8 2.8 16.2 0.5 0.4 0.7 0.4 0.6 12.4 L114 0.8 0.4 3.2 0.3 0.0 3.4 1.01.2 0.4 0.6 0.4 2.9 0.0 2.9 7.2 0.3 1.3 0.9 41.4 3.5 12.9

Substitution of “a Hydrophobic Amino Acid that is Present in aHydrophilic Domain of an α-Helix Structure or a Hydrophilic Amino Acidthat is Present in a Hydrophobic Domain of the Region” to a Residue withHigh Conservation

Mutations were introduced in the same manner as in Example 2 (2) so thatthe above amino acids were respectively substituted to other aminoacids. E. coli BL21 (DE3) was transformed with each of the obtainedmutagenized plasmids, inoculated to LB plates containing ampicillin andcultured at 37° C. for 16 hours. The obtained colonies were cultured in5 mL of LB liquid medium containing IPTG, 0.5 μg/mL of IPTG was added ata cell turbidity of about 0.5 and the cells were cultured at 16° C. for12 hours. After collecting the cells by centrifugation (10,000×g, 2minutes, 4° C.), the cells were resuspended in 250 pt of 20 mM potassiumphosphate buffer (pH 7.0) and a crude enzyme solution was prepared byultrasonication and centrifugation. As a result of analysis of theexpression level in the obtained crude extract by hGH ELISA, expressionwas detected in the group of proteins to which mutations were introducedas shown in FIG. 17. The mutagenized proteins were purified to a singleprotein as shown in FIG. 18 with His GraviTrap (manufactured by GEHealthcare Japan) and it was confirmed that the proteins were solubleproteins. From the above results, it was revealed that the expressionlevel of soluble proteins could be increased by substituting ahydrophobic amino acid that was present in a hydrophilic domain of anα-helix or a hydrophilic amino acid that was present in a hydrophobicdomain of the region to an amino acid with high conservation.

INDUSTRIAL APPLICABILITY

The present invention is useful in the field relating to preparation ofenzyme proteins.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1: Mandelonitrile oxidase gene derived from Chamberliniushualienensis

SEQ ID NO: 2: Amino acid sequence of mandelonitrile oxidase derived fromChamberlinius hualienensis

SEQ ID NO: 3: Amino acid sequence of arginine decarboxylase derived fromArabidopsis thaliana

SEQ ID NO: 4: Arginine decarboxylase gene derived from Arabidopsisthaliana

SEQ ID NO: 5: Amino acid sequence of luciferase (MpLUC) 1-1 derived fromMetridia pacifica

SEQ ID NO: 6: Luciferase (MpLUC) 1-1 gene derived from Metridia pacificaSEQ ID NO: 7: Amino acid sequence of ornithine decarboxylase (DmODC)derived from Drosophila melanogaster

SEQ ID NO: 8: Ornithine decarboxylase (DmODC) gene derived fromDrosophila melanogaster

SEQ ID NO: 9: Amino acid sequence of glutamate dehydrogenase (DmGluDH)derived from Drosophila melanogaster SEQ ID NO: 10: Glutamatedehydrogenase (DmGluDH) gene derived from Drosophila melanogaster

SEQ ID NO: 11: Amino acid sequence of hGH

INCORPORATION OF MATERIAL OF ASCII TEXT SEQUENCE LISTING BY REFERENCE

The material in the ASCII text file sequence listing named,“SIKS1003USDIV_ST25_Corrected_Sequence_Listing” created on Jul. 16,2020, which is 33 kb in size, is hereby incorporated by reference in itsentirety herein.

What is claimed is:
 1. A method for producing an active-form mutantenzyme, comprising steps of: (1) specifying a protein of which a nativeform of the protein exhibits an enzyme activity but which exhibits noenzyme activity or 10% or less enzyme activity of the enzyme activityexhibited by the protein in the native form when a gene of the proteinis expressed in a heterologous expression system to provide aninactive-form enzyme; (2b) determining a sequence conservation insequence identity of at least some amino acid residues in an amino acidsequence of the inactive-form enzyme provided in step (1) and among saidamino acid residues, specifying at least one amino acid residue forwhich sequence conservation of the amino acid in the inactive-formenzyme is lower than sequence conservation of one or more other aminoacids of the same residue; (3b) preparing a gene having a base sequencethat codes for an amino acid sequence which is the amino acid sequenceof the inactive-form enzyme provided in step (1) in which at least onesaid amino acid residue specified in step (2b) is substituted by anotheramino acid of the same residue with a higher sequence conservation thansaid substituted amino acid; and (4b) expressing, in a heterologousexpression system, the gene having the base sequence prepared in step(3b) to obtain a protein that exhibits a same sort of enzyme activity asthe native form of the protein to provide an active form mutant enzyme.2. The method according to claim 1, wherein the amino acid sequence forwhich the sequence conservation in sequence identity is determined instep (2b) is selected from an amino acid sequence of an α-helixstructure region of the inactive-form enzyme provided in step (1). 3.The method according to claim 1, wherein three amino acid residues inwhich sequence conservation of the amino acid in the inactive-formenzyme is lower than sequence conservation of one or more other aminoacids of the same residue are specified in step (2b) and in step 3(b)each of the three amino acid residues specified in step (2b) issubstituted, respectively, by another amino acid of the same residue asthe substituted amino acid that has a higher conservation than saidsubstituted amino acid.
 4. The method according to claim 1, wherein insaid step (3b), the amino acid substitution is carried out for aplurality of said amino acid residues specified in step (2b) and in saidstep (4b), the active-form mutant enzyme that is obtained is a proteinin which a plurality of said amino acid residues specified in step (2b)have been substituted.
 5. The method according to claim 4, whereinsequence conservations of at least two different amino acid sequences ofthe inactive-form enzyme provided in step (1) are determined in step(2b) and in step 3(b) at least one amino acid residue of each of said atleast two different sequences is substituted. respectively, by anotheramino acid of the same residue as the substituted amino acid that has ahigher conservation than said substituted amino acid.
 6. The producingmethod according to claim 1, wherein sequence conservation wascalculated on INTMSAlign by a method comprising by steps of: (i)obtaining BLAST search results by a) entering the sequence of one ormore of α-helix structure regions of the inactive-form enzyme on theBLAST site, b) selecting 5000 as a maximum number of target sequences,ci) using 1.0E⁻³ as Expect threshold, and (ii) comparing proteinsequences obtained from the BLAST search results using the INTMSAlign.7. A method for producing a soluble mutant protein, comprising steps of:(1) specifying a protein of which a native form is a soluble protein butwhich protein is insoluble when a gene of the protein is expressed in aheterologous expression system to provide an insoluble-form protein;(2b) determining a sequence conservation in sequence identity of atleast some amino acid residues in an amino acid sequence of theinsoluble-form protein provided in step (1) and among said amino acidresidues, specifying at least one amino acid residue in which sequenceconservation of the amino acid in the insoluble-form protein is lowerthan sequence conservation of one or more other amino acids of the sameresidue; (3b) preparing a gene having a base sequence that codes for anamino acid sequence which the amino acid sequence of the insoluble-formprotein provided in step (1) in which at least one said amino acidspecified in step (2b) is substituted with another amino acid of thesame amino acid residue with a higher sequence conservation than saidsubstituted amino acid; and (4b) expressing, in a heterologousexpression system, the gene having the base sequence prepared in step(3b) to provide a soluble mutant protein.
 8. The method according toclaim 7, wherein solubility of the soluble mutant protein is determinedbased on an amount of the soluble protein in an extract afterheterologous expression in step (4b).
 9. The method according to claim8, wherein the soluble mutant protein is defined as a mutant protein forwhich the amount of the soluble protein in the extract afterheterologous expression in step (4b) is higher than an amount of solubleprotein in the same extract after heterologous expression of the nativeform protein.
 10. The method according to claim 7, wherein the aminoacid sequence for which the sequence conservation in sequence identityis determined in step (2b) is selected from an amino acid sequence of anα-helix structure region of the insoluble-form protein provided in step(1).
 11. The method according to claim 7, wherein three amino acidresidues in which sequence conservation of the amino acid in theinactive-form enzyme is lower than sequence conservation of one or moreother amino acids of the same residue are specified in step (2b) and instep 3(b) each of the three amino acid residues specified in step (2b)is substituted, respectively, by another amino acid of the same residueas the substituted amino acid that has a higher conservation than saidsubstituted amino acid.
 12. The method according to claim 7, wherein insaid step (3b), the amino acid substitution is carried out for aplurality of said amino acid residues specified in step (2b) and in saidstep (4b), the soluble mutant protein that is obtained is a protein inwhich a plurality of said amino acid residues specified in step (2b)have been substituted.
 13. The method according to claim 12, whereinsequence conservations of at least two different amino acid sequences ofthe insoluble-form protein provided in step (1) are determined in step(2b) and in step (3b) at least amino acid residue of each of said atleast two different sequences is substituted. respectively, by anotheramino acid of the same residue as the substituted amino acid that has ahigher conservation than said substituted amino acid.
 14. The methodaccording to claim 7, wherein sequence conservation was calculated onINTMSAlign by a method comprising by steps of: (i) obtaining BLASTsearch results by a) entering the sequence of one or more of α-helixstructure regions of the inactive-form enzyme on the BLAST site, b)selecting 5000 as a maximum number of target sequences, ci) using 1.0E⁻³as Expect threshold, and (ii) comparing protein sequences obtained fromthe BLAST search results using the INTMSAlign.